U.S. patent application number 10/300683 was filed with the patent office on 2003-12-25 for approaches to identify cystic fibrosis.
Invention is credited to Dunlop, Charles L.M., Weisel, James M..
Application Number | 20030235834 10/300683 |
Document ID | / |
Family ID | 29741002 |
Filed Date | 2003-12-25 |
United States Patent
Application |
20030235834 |
Kind Code |
A1 |
Dunlop, Charles L.M. ; et
al. |
December 25, 2003 |
Approaches to identify cystic fibrosis
Abstract
The present invention relates to the field of genetic screening.
More specifically, the described embodiments concern methods to
screen multiple samples, in a single assay, for the presence or
absence of mutations or polymorphisms in a plurality of genes.
Approaches to screen for the presence or absence of mutations that
are associated with cystic fibrosis and approaches to design
primers that generate extension products that facilitate the
resolution of multiple extension products in a single lane of a gel
or in a single run on a column are also provided.
Inventors: |
Dunlop, Charles L.M.;
(Irvine, CA) ; Weisel, James M.; (Manhatton Beach,
CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
29741002 |
Appl. No.: |
10/300683 |
Filed: |
November 19, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10300683 |
Nov 19, 2002 |
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10142722 |
May 8, 2002 |
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10142722 |
May 8, 2002 |
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PCT/US00/30493 |
Nov 3, 2000 |
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10300683 |
Nov 19, 2002 |
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09851501 |
May 8, 2001 |
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60165301 |
Nov 12, 1999 |
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60333531 |
Nov 19, 2001 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6883 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method of identifying the presence or absence of a genetic
marker in the human cystic fibrosis transmembrane conductance
regulator (CFTR) gene of a subject comprising: providing a DNA
sample from said subject; providing at least one primer set from
Table A; contacting said DNA and said at least one primer set;
generating an extension product from said at least one primer set
that comprises a region of DNA that includes the location of said
genetic marker; separating said extension product on the basis of
melting behavior; and identifying the presence or absence of said
genetic marker in said subject by analyzing the melting behavior of
said extension product.
2. The method of claim 1, wherein at least two primer sets from
Table A are contacted with said DNA.
3. The method of claim 1, wherein at least three primer sets from
Table A are contacted with said DNA.
4. The method of claim 1, wherein at least four primer sets from
Table A are contacted with said DNA.
5. The method of claim 1, wherein at least five primer sets from
Table A are contacted with said DNA.
6. The method of claim 1, wherein at least six primer sets from
Table A are contacted with said DNA.
7. The method of claim 1, wherein at least seven primer sets from
Table A are contacted with said DNA.
8. The method of claim 1, wherein at least eight primer sets from
Table A are contacted with said DNA.
9. The method of claim 2 or 3, wherein the extension products
generated from said primer sets are grouped according to Table E
and separated on the basis of melting behavior.
10. The method of claim 4 or 5, wherein the extension products
generated from said primer sets are grouped according to Table E
and separated on the basis of melting behavior.
11. The method of claim 6 or 7, wherein the extension products
generated from said primer sets are grouped according to Table E
and separated on the basis of melting behavior.
12. The method of claim 8, wherein the extension products generated
from said primer sets are grouped according to Table E and
separated on the basis of melting behavior.
13. A method of identifying the presence or absence of a genetic
marker in the human cystic fibrosis transmembrane conductance
regulator (CFTR) gene of a subject comprising: providing a DNA
sample from said subject; providing at least one primer set from
Table 2; contacting said DNA and said at least one primer set;
generating an extension product from said at least one primer set
that comprises a region of DNA that includes the location of said
genetic marker; separating said extension product on the basis of
melting behavior; and identifying the presence or absence of said
genetic marker in said subject by analyzing the melting behavior of
said extension product.
14. The method of claim 13, wherein at least two primer sets from
Table 2 are contacted with said DNA.
15. The method of claim 13, wherein at least three primer sets from
Table 2 are contacted with said DNA.
16. The method of claim 13, wherein at least four primer sets from
Table 2 are contacted with said DNA.
17. The method of claim 13, wherein at least five primer sets from
Table 2 are contacted with said DNA.
18. The method of claim 13, wherein at least six primer sets from
Table 2 are contacted with said DNA.
19. The method of claim 13, wherein at least seven primer sets from
Table 2 are contacted with said DNA.
20. The method of claim 13, wherein at least eight primer sets from
Table 2 are contacted with said DNA.
21. The method of claim 14 or 15, wherein the extension products
generated from said primer sets are grouped according to Table 3
and separated on the basis of melting behavior.
22. The method of claim 16 or 17, wherein the extension products
generated from said primer sets are grouped according to Table 3
and separated on the basis of melting behavior.
23. The method of claim 18 or 19, wherein the extension products
generated from said primer sets are grouped according to Table 3
and separated on the basis of melting behavior.
24. The method of claim 20, wherein the extension products
generated from said primer sets are grouped according to Table 3
and separated on the basis of melting behavior.
25. A method of identifying the presence or absence of a genetic
marker in the human cystic fibrosis transmembrane conductance
regulator (CFTR) gene of a subject comprising: providing a DNA
sample from said subject; providing at least one primer set that is
any number between 1-75 nucleotides upstream or downstream of a
primer set from Table A; contacting said DNA and said at least one
primer set; generating an extension product from said at least one
primer set that comprises a region of DNA that includes the
location of said genetic marker; separating said extension product
on the basis of melting behavior; and identifying the presence or
absence of said genetic marker in said subject by analyzing the
melting behavior of said extension product.
26. The method of claim 25, wherein at least two primer sets that
are any number between 1-75 nucleotides upstream or downstream of a
primer set from Table A are contacted with said DNA.
27. The method of claim 25, wherein at least three primer sets that
are any number between 1-75 nucleotides upstream or downstream of a
primer set from Table A are contacted with said DNA.
28. The method of claim 25, wherein at least four primer sets that
are any number between 1-75 nucleotides upstream or downstream of a
primer set from Table A are contacted with said DNA.
29. The method of claim 25, wherein at least five primer sets that
are any number between 1-75 nucleotides upstream or downstream of a
primer set from Table A are contacted with said DNA.
30. The method of claim 25, wherein at least six primer sets that
are any number between 1-75 nucleotides upstream or downstream of a
primer set from Table A are contacted with said DNA.
31. The method of claim 25, wherein at least seven primer sets that
are any number between 1-75 nucleotides upstream or downstream of a
primer set from Table A are contacted with said DNA.
32. The method of claim 25, wherein at least eight primer sets that
are any number between 1-75 nucleotides upstream or downstream of a
primer set from Table A are contacted with said DNA.
33. The method of claim 26 or 27, wherein the extension products
generated from said primer sets are grouped according to Table E
and separated on the basis of melting behavior.
34. The method of claim 28 or 29, wherein the extension products
generated from said primer sets are grouped according to Table E
and separated on the basis of melting behavior.
35. The method of claim 30 or 31, wherein the extension products
generated from said primer sets are grouped according to Table E
and separated on the basis of melting behavior.
36. The method of claim 32, wherein the extension products
generated from said primer sets are grouped according to Table E
and separated on the basis of melting behavior.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/333531, filed Nov. 19, 2002, which is hereby
expressly incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of genetic
screening. More specifically, the described embodiments concern
methods to screen multiple samples, in a single assay, for the
presence or absence of mutations or polymorphisms in a plurality of
genes. Approaches to screen for the presence or absence of
mutations that are associated with cystic fibrosis and approaches
to design primers that generate extension products that facilitate
the resolution of multiple extension products in a single lane of a
gel or in a single run on a column are also provided.
BACKGROUND OF THE INVENTION
[0003] Despite the tremendous progress in molecular biology and the
identification of genes, mutations, and polymorphisms responsible
for disease, the ability to rapidly screen a subject for the
presence of multiple disorders has been technically difficult and
cost prohibitive. Current DNA-based diagnostics allow for the
identification of a single mutation or polymorphism or gene per
analysis. Although high-throughput methods and gene chip technology
have enabled the ability to screen multiple samples or multiple
loci within the same sample, these approaches require several
independent reactions, which increases the time required to process
clinical samples and drastically increases the cost. Further,
because of time and expense, conventional diagnostic approaches
focus on the identification of the presence of DNA fragments that
are associated with a high frequency of mutation, leaving out
analysis of other loci that may be critical to diagnose a disease.
The need for a better way to diagnose genetic disease is
manifest.
[0004] With the advent of multiplex Polymerase Chain Reaction
(PCR), the ability to use multiple primer sets to generate multiple
extension products from a single gene is at hand. By hybridizing
isolated DNA with multiple sets of primers that flank loci of
interest on a single gene, it is possible to generate a plurality
of extension products in a single PCR reaction corresponding to
fragments of the gene. As the number of primers increases, however,
the complexity of the reaction increases and the ability to resolve
the extension products using conventional techniques fails.
Further, since many diseases are caused by changes of a single
nucleotide, the rapid detection of the presence or absence of these
mutations or polymorphisms is frustrated by the fact that the PCR
products that indicate both the diseased and non-diseased state are
of the same size.
[0005] Developments in gel electrophoresis and high performance
liquid chromatography (HPLC), however, have enabled the separation
of double-stranded DNAs based upon differences in their melting
behaviors, which has allowed investigators to resolve DNA fragments
having a single mutation or single polymorphism. Techniques such as
temporal temperature gradient gel electrophoresis (TTGE) and
denaturing high performance liquid chromatography (DHPLC) have been
used to screen for small changes or point mutations in DNA
fragments.
[0006] The separation principle of TTGE, for example, is based on
the melting behavior of DNA molecules. In a denaturing
polyacrylamide gel, double-stranded DNA is subject to conditions
that will cause it to melt in discrete segments called "melting
domains." The melting temperature T.sub.m of these domains is
sequence-specific. When the T.sub.m of the lowest melting domain is
reached, the DNA will become partially melted, creating branched
molecules. Partial melting of the DNA reduces its mobility in a
polyacrylamide gel.
[0007] Since the T.sub.m of a particular melting domain is
sequence-specific, the presence of a mutation or polymorphism will
alter the melting profile of that DNA in comparison to the
wild-type or non-polymorphic DNA. That is, a heteroduplex DNA
consisting of a wild-type or non-polymorphic strand annealed to
mutant or poymorphic strand, will melt at a lower temperature than
a homoduplex DNA strand consisting of two wild-type or
non-polymorphic strands. Accordingly, the DNA containing the
mutation or polymorphism will have a different mobility compared to
the wild-type or non-polymorphic DNA. The TTGE approach has been
used as a method for screening for mutations in the cystic fibrosis
gene, for example. (Bio-Rad U.S./E.G. Bulletin 2103, herein
expressly incorporated by reference in its entirety).
[0008] Similarly, the separation principle of DHPLC is based on the
melting or denaturing behavior of DNA molecules. As the use and
understanding of HPLC developed, it became apparent that when HPLC
analyses were carried out at a partially denaturing temperature,
i.e., a temperature sufficient to denature a heteroduplex at the
site of base pair mismatch, homoduplexes could be separated from
heteroduplexes having the same base pair length. (See e.g.,
Hayward-Lester, et al., Genome Research 5:494 (1995); Underhill, et
al., Proc. Natl. Acad. Sci. USA 93:193 (1996); Oefner, et al.,
DHPLC Workshop, Stanford University, Palo Alto, Calif., (Mar. 17,
1997); Underhill, et al., Genome Research 7:996 (1997); Liu, et
al., Nucleic Acid Res., 26:1396 (1998), all of which and the
references contained therein are hereby expressly incorporated by
reference in their entireties). Techniques such as Matched Ion
Polynucleotide Chromatography (MIPC) and Denaturing Matched Ion
Polynucleotide Chromatography (DMIPC) have also been employed to
increase the sensitivity of detection. It was soon realized that
DHPLC, which for the purposes of this disclosure includes but is
not limited to, MIPC, DMIPC, and ion-pair reverse phase
high-performance liquid chromatography, could be used to separate
heteroduplexes from homoduplexes that differed by as little as one
base pair. Various DHPLC techniques have been described in U.S.
Pat. Nos. 5,795,976; 5,585,236; 6,024,878; 6,210,885; Huber, et
al., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem.
212:351 (1993); Huber, et al., Anal. Chem. 67:578 (1995); O'Donovan
et al., Genomics 52:44 (1998), Am J Hum Genet.
December;67(6):1428-36 (2000); Ann Hum Genet. September:63 (Pt
5):383-91 (1999); Biotechniques, April;28(4):740-5 (2000);
Biotechniques. November;29(5):1084-90, 1092 (2000); Clin Chem.
August;45(8 Pt 1):1133-40 (1999); Clin Chem. April;47(4):635-44
(2001); Genomics. August 15;52(1):44-9 (1998); Genomics. March
15;56(3):247-53 (1999); Genet Test. ;1(4):237-42 (1997-98); Genet
Test.:4(2):125-9 (2000); Hum Genet. June; 106(6):663-8(2000); Hum
Genet. Nov;107(5):483-7 (2000); Hum Genet. November;107(5):488-93
(2000); Hum Mutat. December;16(6):518-26 (2000); Hum Mutat.
15(6):556-64 (2000); Hum Mutat. March ;17(3):210-9 (2001); J
Biochem Biophys Methods. November 20;46(1-2):83-93 (2000); J
Biochem Biophys Methods. January 30;47(1-2):5-19 (2001); Mutat Res.
November 29;430(1):13-21(1999); Nucleic Acids Res. March
1;28(5):E13 (2000); and Nucleic Acids Res. October 15;28(20):E89
(2000), all of which, including the references contained therein,
are hereby expressly incorporated by reference in their entireties.
Despite the efforts of many, there remains a need for a better
approach to screen for mutations and/or polymorphisms.
BRIEF SUMMARY OF THE INVENTION
[0009] Aspects of the invention concern rapid and inexpensive but
efficient approaches to determine the presence or absence of
genetic markers associated with cystic fibrosis. Several
oligonucleotide primers specific for the human cystic fibrosis
transmembrane conductance regulator (CFTR) gene have been developed
(e.g., TABLE A and TABLE 2). These primers and oligonucleotides
that are any number between 1-75 nucleotides upstream or downstream
of said primers are unique in sequence and in their ability to
generate extension products that melt evenly over vast stretches of
nucleotides, which greatly improves the sensitivity of detection
(e.g., single base mutations). It was then realized that by
grouping extension products with similar melting behaviors, one can
rapidly and efficiently separate multiple extension products on the
basis of melting behavior on the same lane of a TTGE gel or in the
same run on a DHPLC. Accordingly, a rapid, inexpensive and
efficient approach to diagnose a subject suffering from cystic
fibrosis or a carrier of the disease was discovered, whereby
extension products are generated from a subject's DNA using the
primers described herein, the extension products are grouped or
mixed according to their melting behavior, and the grouped or mixed
extension products are separated on the basis of melting behavior
(e.g., one group per lane of TTGE gel). Not only does the pooling
of extension products reduce cost and the time to perform the
analysis but, because the extension products are optimized for
melting behavior, the sensitivity of detection remains very
high.
[0010] By one approach, for example, a method of identifying the
presence or absence of a genetic marker in the human cystic
fibrosis transmembrane conductance regulator (CFTR) gene of a
subject is conducted by providing a DNA sample from said subject;
providing at least one primer set from TABLE A; contacting said DNA
and said at least one primer set; generating an extension product
from said at least one primer set that comprises a region of DNA
that includes the location of said genetic marker; separating said
extension product on the basis of melting behavior; and identifying
the presence or absence of said genetic marker in said subject by
analyzing the melting behavior of said extension product. In
related embodiments, at least 2, 3, 4, 5, 6, 7, or 8 primer sets
from TABLE A are contacted with said DNA. In more related
embodiments, the extension products generated from said 2, 3, 4, 5,
6, 7, or 8 primer sets are grouped according to TABLE E and
separated on the basis of melting behavior.
[0011] By another approach, a method of identifying the presence or
absence of a genetic marker in the human cystic fibrosis
transmembrane conductance regulator (CFTR) gene of a subject is
conducted by providing a DNA sample from said subject; providing at
least one primer set from TABLE 2; contacting said DNA and said at
least one primer set; generating an extension product from said at
least one primer set that comprises a region of DNA that includes
the location of said genetic marker; separating said extension
product on the basis of melting behavior; and identifying the
presence or absence of said genetic marker in said subject by
analyzing the melting behavior of said extension product. In
related embodiments, at least 2, 3, 4, 5, 6, 7, or 8 primer sets
from TABLE 2 are contacted with said DNA. In more related
embodiments, the extension products generated from said 2, 3, 4, 5,
6, 7, or 8 primer sets are grouped according to TABLE 3 and
separated on the basis of melting behavior.
[0012] In another set of embodiments, a method of identifying the
presence or absence of a genetic marker in the human cystic
fibrosis transmembrane conductance regulator (CFTR) gene of a
subject is conducted by providing a DNA sample from said subject;
providing at least one primer set that is any number between 1-75
nucleotides upstream or downstream of a primer set from TABLE A;
contacting said DNA and said at least one primer set; generating an
extension product from said at least one primer set that comprises
a region of DNA that includes the location of said genetic marker;
separating said extension product on the basis of melting behavior;
and identifying the presence or absence of said genetic marker in
said subject by analyzing the melting behavior of said extension
product. In related embodiments, at least 2, 3, 4, 5, 6, 7, or 8
primer sets from TABLE A are contacted with said DNA. In more
related embodiments, the extension products generated from said 2,
3, 4, 5, 6, 7, or 8 primer sets are grouped according to TABLE E
and separated on the basis of melting behavior.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows a melting curve for the extension product CF3A
spanning nucleotides 112-275 of the human cystic fibrosis
transmembrane conductance regulator (CFTR) gene. The x axis shows
the number of nucleotides and the y axis shows the temperature.
[0014] FIG. 2 shows a melting curve for the extension product CF3B
spanning nucleotides 215-300 of the CFTR gene. The x axis shows the
number of nucleotides and the y axis shows the temperature.
[0015] FIG. 3 shows a melting curve for the extension product
CFTR1B spanning nucleotides 228-323 of the CFTR gene. The x axis
shows the number of nucleotides and the y axis shows the
temperature.
[0016] FIG. 4 shows a melting curve for the extension product
CFTR1A spanning nucleotides 123-230 of the CFTR gene. The x axis
shows the number of nucleotides and the y axis shows the
temperature.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Embodiments described herein concern a novel approach to
screen for the presence or absence of multiple mutations or
polymorphisms in a plurality of genes, thus, improving the speed
and lowering the cost to diagnose genetic diseases. Particularly
preferred embodiments concern approaches to screen multiple loci in
the human cystic fibrosis transmembrane conductance regulator
(CFTR) gene so as to determine a cystic fibrosis carrier status or
diagnose the disease. Several embodiments also permit very
sensitive detection of single base mutations, single base
mismatches, and small nuclear polymorphisms (SNPs), as well as,
larger alterations in DNA at multiple loci, in a plurality of
genes, in multiple samples. Further, by employing a DNA standard or
by screening a plurality of DNA samples in the same assay, improved
sensitivity of detection can be obtained. A novel approach to
designing primers and extension products generated therefrom is
described in the context of an assay that was performed to detect
the presence or absence of genetic markers, polymorphisms, or
mutations on the Cystic Fibrosis Transmembrane Conductance
Regulator gene (CFTR).
[0018] Embodiments include methods of identifying the presence or
absence of a plurality of genetic markers in a subject in the same
gene or separate genes. One method is practiced, for example, by
providing a DNA sample from said subject, providing a plurality of
nucleic acid primer sets that hybridize to said DNA at regions that
flank said plurality of genetic markers, wherein each primer set
has a first and a second primer and, wherein said plurality of
genetic markers exist on the same gene or a plurality of genes,
contacting said DNA and said plurality of nucleic acid primer sets
in a single reaction vessel or multiple reaction vessels,
generating, in said reaction vessel(s), a plurality of extension
products that comprise regions of DNA that include the location of
said plurality of genetic markers, separating said plurality of
extension products on the basis of melting behavior in a single
lane or multiple lanes of a gel or a single run or multiple runs on
a column, and identifying the presence or absence of said plurality
of genetic markers in said subject by analyzing the melting
behavior of said plurality of extension products. In some aspects
of this method the separation on the basis of melting behavior is
accomplished by TTGE and in other embodiments the separation on the
basis of melting behavior is accomplished by DHPLC. In some
embodiments, said extension products are first separated by size
for a period sufficient to separate populations of extension
products and then separated by melting behavior. The size
separation can be accomplished on the TTGE gel or DHPLC column
prior to separating on the basis of melting behavior.
[0019] Preferably, after generating the extension products by an
amplification technique (e.g., Polymerase Chain Reaction or PCR),
the extension products are grouped and pooled according to their
predicted and/or actual melting behavior. In this way, multiple
extension products, which correspond to different regions on the
same gene or different regions on a plurality of genes can be
separated on the same lane of a TTGE gel or in the same run on a
DHPLC column. By carefully designing the primers, such that the
extension products generated therefrom melt over large stretches of
DNA (approximately 25, 50, 75, 100, 125, or 150 nucleotides) at
roughly the same temperature (within up to 1.5.degree. C. of one
another), it was unexpectedly discovered that multiple extension
products (2, 3, 4, 5, 6 or more) can be separated on the same lane
of a TTGE gel or in the same run on an DHPLC column, thereby
substantially reducing the cost of conducting the analysis.
[0020] In some embodiments, the subject is selected from the group
consisting of a plant, virus, bacteria, mold, yeast, animal, and
human and either the first or the second primer comprise a GC
clamp. In other aspects of the invention, either the first or the
second primer hybridize to a sequence within an intron. Preferably,
at least one of the plurality of genetic markers is indicative of a
disease selected from the group consisting of familial
hypercholesterolemia (FH), cystic fibrosis, Tay-sachs, thalassemia,
sickle cell disease, phenylketonuria, galactosemia, fragile X
syndrome, hemophilia A, myotonic dystrophy, medium-chain acyl CoA
dehydrogenase, maturity onset diabetes, cystinuria, methylmolonic
acidemia, urea cycle disorders, hereditary fructose intolerance,
hereditary hemachromatosis, neonatal thrombocytopenia, Gaucher's
disease, tyrosinemia, Wilson's disease, alcaptonuria, hypolactasia,
Baker's disease, argininemia Adenomatous polyposis coli (APC),
Adult Polycystic Kidney disease, a-1-antitrypsin deficiency,
Duchenne Muscular Dystrophy, Hemophilia A, Hereditary Nonpolyposis
colorectal cancer, Huntingtons disease, Marfans syndrome, Myotonic
dystrophy, Neurofibromatosis, Osteogenesis imperfecta,
Retinoblastoma, Sickle cell disease, Freidrichs ataxia,
Hemoglobinopathies, Leber's hereditary optic neuropathy, MCAD,
Canavan's disease, Retintitus Pigmentosa, Bloom Syndrome, Fanconi
anemia, and Neimann Pick disease.
[0021] In other embodiments, the plurality of primer sets consist
of at least 3, 4, 5, 6, or 7 primer sets. Additionally, in some
embodiments, the plurality of genes consist of at least 2, 3, 4, 5,
6, or 7 genes. The method above preferably generates the extension
products using the Polymerase Chain Reaction (PCR) and the method
can be supplemented by a step in which a control DNA is added.
[0022] Another embodiment concerns a method of identifying the
presence or absence of a plurality of genetic markers in a
plurality of subjects. This method is practiced by: providing a DNA
sample from said plurality of subjects, providing a plurality of
nucleic acid primer sets that hybridize to said DNA at regions that
flank said plurality of genetic markers, wherein each primer set
has a first and a second primer and, wherein said plurality of
genetic markers exist on the same gene or on a plurality of genes,
contacting said DNA and said plurality of nucleic acid primer sets
in a single reaction vessel or multiple vessels, generating, in
said reaction vessel(s), a plurality of extension products that
comprise regions of DNA that include the location of said plurality
of genetic markers, separating said plurality of extension products
on the basis of melting behavior in a single lane or multiple lanes
of a gel or a single run or multiple runs on a column, and
identifying the presence or absence of said plurality of genetic
markers in said plurality of subjects by analyzing the melting
behavior of said plurality of extension products. In some aspects
of this embodiment, the separation on the basis of melting behavior
is accomplished by TTGE and in other embodiments the separation on
the basis of melting behavior is accomplished by DHPLC.
[0023] As above, preferably, after generating the extension
products by the amplification technique (e.g., PCR) from the
plurality of subjects, the extension products are grouped and
pooled according to their predicted and/or actual melting behavior.
By separating multiple extension products generated from a
plurality of subjects in the same lane of a TTGE gel or in the same
run on a DHPLC column, the cost of analysis is substantially
reduced. Because the incidence of polymorphism or mutation in the
population as a whole is small, the large scale screening,
described above, can be performed. When a polymorphism and/or
mutation is detected in this type of assay, single subject assays
can be performed, as described above, to identify the subject(s)
that have the polymorphism and/or mutation.
[0024] In other embodiments, the subject is selected from the group
consisting of a plant, virus, bacteria, mold, yeast, animal, and
human and either the first or the second primer comprise a GC
clamp. In other aspects of this embodiment, either the first or the
second primer hybridize to a sequence within an intron. Preferably,
at least one of the plurality of genetic markers is indicative of a
disease selected from the group consisting of familial
hypercholesterolemia (FH), cystic fibrosis, Tay-sachs, thalassemia,
sickle cell disease, phenylketonuria, galactosemia, fragile X
syndrome, hemophilia A, myotonic dystrophy, medium-chain acyl CoA
dehydrogenase, maturity onset diabetes, cystinuria, methylmolonic
acidemia, urea cycle disorders, hereditary fructose intolerance,
hereditary hemachromatosis, neonatal thrombocytopenia, Gaucher's
disease, tyrosinemia, Wilson's disease, alcaptonuria, hypolactasia,
Baker's disease, argininemia Adenomatous polyposis coli (APC),
Adult Polycystic Kidney disease, a-1-antitrypsin deficiency,
Duchenne Muscular Dystrophy, Hemophilia A, Hereditary Nonpolyposis
colorectal cancer, Huntingtons disease, Marfans syndrome, Myotonic
dystrophy, Neurofibromatosis, Osteogenesis imperfecta,
Retinoblastoma, Sickle cell disease, Freidrichs ataxia,
Hemoglobinopathies, Leber's hereditary optic neuropathy, MCAD,
Canavan's disease, Retintitus Pigmentosa, Bloom Syndrome, Fanconi
anemia, and Neimann Pick disease.
[0025] In more embodiments, the plurality of subjects consist of at
least 2, 3, 4, 5, 6, or 7 subjects. In more aspects of this
embodiment, the plurality of primer sets consist of at least 3, 4,
5, 6, or 7 primer sets. Additionally, in some embodiments, the
plurality of genes consist of at least 2, 3, 4, 5, 6, or 7 genes.
The method above preferably generates the extension products using
PCR and the method can be supplemented by a step in which a control
DNA is added.
[0026] Still another embodiment involves a method of identifying
the presence or absence of a mutation or polymorphism in a subject.
This method is practiced by: providing a DNA sample from said
subject, generating a population of extension products from said
sample, wherein said extension products comprise a region of said
DNA that corresponds to the location of said mutation or
polymorphism, providing at least one control DNA, wherein said
control DNA corresponds to the extension product but lacks said
mutation or polymorphism, contacting said control DNA and said
population of extension products in a single reaction vessel,
thereby forming a mixed DNA sample, heating said mixed DNA sample
to a temperature sufficient to denature said control DNA and said
DNA sample, cooling said mixed DNA sample to a temperature
sufficient to anneal said control DNA and said DNA sample,
separating said mixed sample on the basis of melting behavior in a
single lane or multiple lanes of a gel or a single run or multiple
runs on a column, and identifying the presence or absence of said
mutation or polymorphism by analyzing the melting behavior of said
mixed DNA sample. By this approach, the addition of the control DNA
followed by the heating and cooling steps, forces heteroduplex
formation, if a polymorphism or mutation is present, which
facilitates identification. In some aspects of this embodiment, the
control DNA is DNA obtained or amplified from a second subject and
the presence or absence of a mutation or polymorphism is known. In
other aspects of the invention, heteroduplex formation can be
forced by pooling the extension products generated from a plurality
of subjects and denaturing and annealing, as above. Because the
predominant genotype in a plurality of subjects lacks polymorphisms
or mutations in the gene(s) analyzed, the majority of the DNA will
force heteroduplex formation with any polymorphic or mutant DNA in
the pool. Accordingly, the identification of mutant and/or
polymorphic DNA is facilitated and the cost of the analysis is
reduced. In some aspects of this embodiment, the separation on the
basis of melting behavior is accomplished by TTGE and in other
embodiments the separation on the basis of melting behavior is
accomplished by DHPLC.
[0027] Still more embodiments concern the primers or groups of
primers disclosed herein (preferably CFTR specific primers), kits
containing said nucleic acids, and methods of using these primers
or groups of primers to diagnose a carrier status or the presence
of disease (e.g., cystic fibrosis). These nucleic acid primers can
be used to efficiently determine the presence or absence of a
polymorphism or mutation in a multiplex PCR reaction that screens a
plurality of genes and a plurality of subjects in a single reaction
vessel or multiple reaction vessels. Additionally, reaction vessels
comprising a DNA sample, and a plurality of nucleic acid primer
sets that hybridize to said DNA sample at regions that flank a
plurality of genetic markers, wherein said plurality of genetic
markers exist on a single gene or a plurality of genes are
embodiments. Further, a reaction vessel comprising a plurality of
DNA samples obtained from a plurality of subjects and a plurality
of nucleic acid primer sets that hybridize to said plurality of DNA
samples at regions that flank a plurality of genetic markers,
wherein said plurality of genetic markers exist on a plurality of
genes or on a single gene are embodiments. Still more aspects of
the invention include a reaction vessel containing a plurality of
extension products (2, 3, 4, 5, 6, 7, 8, 9, or 10 or more), which
melt at approximately the same temperature (e.g., 0.degree.
C.-1.5.degree. C. from one another).
[0028] Other embodiments concern a gel having lanes and adapted to
separate different DNAs comprising a plurality of extension
products, in a single lane of said gel, wherein said plurality of
extension products melt at approximately the same temperature but
are resolvable on said gel and, which correspond to regions of DNA
located on a plurality of genes or on a single gene and, wherein
said regions of DNA comprise loci that indicate a genetic trait and
a gel having lanes and adapted to separate different DNAs
comprising a plurality of extension products, in a single lane of
said gel, wherein said plurality of extension products correspond
to regions of DNA located on a plurality of genes or on a single
gene in a single individual or a plurality of subjects and, wherein
said regions of DNA comprise loci that indicate a genetic
trait.
[0029] Additional embodiments include a DHPLC column adapted to
separate different DNAs comprising a plurality of extension
products, wherein said plurality of extension products melt at
approximately the same temperature but are resolvable on said
column and, which correspond to regions of DNA located on a
plurality of genes or a single gene or and, wherein said regions of
DNA comprise loci that indicate a genetic trait and a DHPLC column
adapted to separate different DNAs comprising a plurality of
extension products, wherein said plurality of extension products
correspond to regions of DNA located on a plurality of genes or on
a single gene in a single individual or a plurality of subjects
and, wherein said regions of DNA comprise loci that indicate a
genetic trait. More description of the compositions and methods
described above is provided in the in the following sections.
[0030] Approaches to Facilitate and Reduce the Cost of Genetic
Analysis
[0031] Aspects of the invention described herein concern approaches
to analyze DNA samples for the presence or absence of a plurality
of genetic markers that reside on a plurality of genes in a single
assay. Some embodiments allow one to rapidly distinguish a
plurality of DNA fragments in a single sample that differ only
slightly in size and/or composition (e.g., a single base change,
mutation, or polymorphism). Other embodiments concern methods to
screen multiple genes from a subject, in a single assay, for the
presence or absence of a mutation or polymorphism. An approach to
achieve greater sensitivity of detection of mutations or
polymorphisms present in a DNA sample is also provided. Preferred
embodiments, however, include methods to screen multiple genes, in
a plurality of DNA samples, in a single assay, for the presence or
absence of mutations or polymorphisms.
[0032] It was discovered that multiple extension products that have
slight differences in length and/or composition can be resolved by
separating the DNA on the basis of melting temperature. By one
approach, a plurality of varying lengths of double-stranded DNA are
applied to a denaturing gel and the double-stranded DNAs are
separated by applying an electrical current while the temperature
of the gel is raised gradually. By slowly increasing the
temperature while the DNA is electrically separated on a
polyacrylamide gel containing a denaturant (e.g., urea), the dsDNA
eventually denatures to partially single stranded (branched
molecules) DNA. Because branched or heteroduplex DNA migrates more
rapidly or more slowly than dsDNA or homoduplex DNA, one can
quickly determine the differences in melting behavior between DNA
fragments, compare this melting temperature to a standard DNA
(e.g., a wild-type DNA or non-polymorphic DNA), and identify the
presence or absence of a mutation or polymorphism in the screened
DNA. This technique efficiently separates multiple DNA fragments,
generated by a single multiplex PCR reaction on a plurality of loci
from different genes (e.g., in one experiment, 10 different loci
were analyzed in the same reaction and each of the extension
products, some that differed by only a single mutation, were
efficiently resolved).
[0033] It was also discovered that multiple extension products that
have slight differences in length and/or composition can be
resolved by separating the DNA by DHPLC. By one approach, a
plurality of varying lengths of double-stranded DNA are applied to
a ion-pair reverse phase HPLC column (e.g., alkylated non-porous
poly(styrene-divinylbenzene))that has been equilibrated to an
appropriate denaturing temperature, depending on the size and
composition of the DNA to be separated (e.g., 53.degree. C. to
63.degree. C.) in an appropriate buffer (e.g., 0.1 mM triethylamine
acetate (TEAA) pH 7.0). Once applied to the column, the double
stranded DNA binds to the matrix. By slowly increasing the presence
of a denaturant (e.g., acetonitrile in TEAA), the dsDNA eventually
denatures to partially single stranded (branched molecules) DNA and
elutes from the column. Preferably a linear gradient is used to
slowly elute the bound DNA. Detection can be accomplished using a
U.V. detector, radioactivity, dyes, or fluoresence. In some
embodiments, the extension products are first separated on the
basis of size using a shallow gradient of denaturant for a time
sufficient to separate individual populations of extension products
and then on the basis of melting behavior using a deeper gradient
of denaturant. The techniques described in the following references
can also be modified for use with aspects of the invention: U.S.
Pat. Nos. 5,795,976; 5,585,236; 6,024,878; 6,210,885; Huber, et
al., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem.
212:351 (1993); Huber, et al., Anal. Chem. 67:578 (1995); O'Donovan
et al., Genomics 52:44 (1998), Am J Hum Genet.
December;67(6):1428-36 (2000); Ann Hum Genet. September:63 (Pt
5):383-91 (1999); Biotechniques, April;28(4):740-5 (2000);
Biotechniques. November;29(5):1084-90, 1092 (2000); Clin Chem.
August;45(8 Pt 1):1133-40 (1999); Clin Chem. April;47(4):635-44
(2001); Genomics. August 15;52(1):44-9 (1998); Genomics. March
15;56(3):247-53 (1999); Genet Test. ; 1(4):237-42 (1997-98); Genet
Test.:4(2):125-9 (2000); Hum Genet. Jun;106(6):663-8 (2000); Hum
Genet. November;107(5):483-7 (2000); Hum Genet.
November;107(5):488-93 (2000); Hum Mutat. December;16(6):518-26
(2000); Hum Mutat. 15(6):556-64 (2000); Hum Mutat.
March;17(3):210-9 (2001); J Biochem Biophys Methods. November
20;46(1-2):83-93 (2000); J Biochem Biophys Methods. January
30;47(1-2):5-19 (2001); Mutat Res. November 29;430(l):13-21(1999);
Nucleic Acids Res. March 1;28(5):E13 (2000); and Nucleic Acids Res.
October 15;28(20):E89 (2000), all of which are hereby expressly
incorporated by reference in their entireties including the
references cited therein,
[0034] Because branched or heteroduplex DNA elutes either more
rapidly or more slowly than homoduplex DNA, one can quickly
determine the differences in melting behavior between DNA
fragments, compare this melting temperature to a standard DNA
(e.g., a wild-type or non-polymorphic homoduplex DNA), and identify
the presence or absence of a mutation or polymorphism in the
screened DNA. This technique efficiently separates multiple DNA
fragments, generated by a single multiplex PCR reaction on a
plurality of loci from different genes.
[0035] Some of the embodiments described herein have adapted the
DNA separation techniques described above to allow for
high-throughput genetic screening of organisms (e.g., plant, virus,
bacteria, mold, yeast, and animals including humans). Typically,
multiple primers that flank genetic markers (e.g., mutations or
polymorphisms that indicate a congenital disease or a trait) on
different genes are employed in a single amplification reaction and
the multiple extension products are separated on a denaturing gel
or by DHPLC according to their melting behavior. The presence or
absence of mutations or polymorphisms, also referred to as "genetic
markers", in the subject's DNA are then detected by identifying an
aberrant melting behavior in the extension products (e.g.,
migration on a gel that is too fast or too slow or elution from a
DHPLC column that is too fast or too slow). Advantageously, some
embodiments provide a greater understanding of a subject's health
because more loci that are indicative of disease, for example, are
analyzed in a single assay. Further, some embodiments drastically
reduce the cost of performing such diagnostic assays because many
different genes and markers for disease can be screened
simultaneously in a single assay.
[0036] By one approach, for example, a biological sample from the
subject (e.g., blood) is obtained by conventional means and the DNA
is isolated. Next, the DNA is hybridized with a plurality of
nucleic acid primers that flank regions of a plurality of genetic
loci or markers that are associated with or linked to the plurality
of traits to be analyzed. Although 10 different loci have been
detected in a single assay (requiring 20 primers), more or less
loci can be screened in a single assay depending on the needs of
the user. Preferably, each assay has sufficient primers to screen
at least three different loci, which may be located on three
different genes. That is, the embodied assays can employ sufficient
primers to screen at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 24 or more, independent loci or markers
that are indicative of a disease in a single assay and these loci
can be on different genes. Because more than one loci or marker can
be detected by a single set of primers, the detection of 20
different markers, for example, can be accomplished with less than
40 primers. However, in many assays, a different set of primers is
needed to detect each different loci. Thus, in several embodiments,
at least 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, or more primers are used.
[0037] Desirably, the primers hybridize to regions of human DNA
that flank markers or loci associated with or linked to human
diseases such as: familial hypercholesterolemia (FH), cystic
fibrosis, Tay-sachs, thalassemia, sickle cell disease,
phenylketonuria, galactosemia, fragile X syndrome, hemophilia A,
myotonic dystrophy, medium-chain acyl CoA dehydrogenase, maturity
onset diabetes, cystinuria, methylmolonic acidemia, urea cycle
disorders, hereditary fructose intolerance, hereditary
hemachromatosis, neonatal thrombocytopenia, Gaucher's disease,
tyrosinemia, Wilson's disease, alcaptonuria, hypolactasia, Baker's
disease, argininemia Adenomatous polyposis coli (APC), Adult
Polycystic Kidney disease, a-1-antitrypsin deficiency, Duchenne
Muscular Dystrophy, Hemophilia A, Hereditary Nonpolyposis
colorectal cancer, Huntingtons disease, Marfans syndrome, Myotonic
dystrophy, Neurofibromatosis, Osteogenesis imperfecta,
Retinoblastoma, Sickle cell disease, Freidrichs ataxia,
Hemoglobinopathies, Leber's hereditary optic neuropathy, MCAD,
Canavan's disease, Retintitus Pigmentosa, Bloom Syndrome, Fanconi
anemia, and Neimann Pick disease. It should be understood, however,
that the list above is not intended to limit the invention in any
way and the techniques described herein can be used to detect and
identify any gene or mutation or polymorphism desired (e.g.,
polymorphisms or mutations associated with alcohol dependence,
obesity, and cancer).
[0038] Once the primers are hybridized to the subject's DNA; a
plurality of extension products having the marker or loci
indicative of the trait are generated. Preferably, the extension
products are generated through a polymerase-driven amplification
reaction, such as multiplex PCR or multiplex Ligase Chain Reaction
(LCR). Then the extension products are separated on the basis of
melting behavior (e.g., TTGE or DHPLC).
[0039] In some approaches, for example, the extension products are
isolated from the reactants in the amplification reaction,
suspended in a non-denaturing loading buffer, and are loaded on a
TTGE denaturing gel (e.g., an 8%, 7M urea polyacrylamide gel). The
sample can be heated to a temperature sufficient to denature a DNA
duplex and then cooled to a temperature that allows reannealing,
prior to suspending the DNA in the non-denaturing loading buffer.
The extension products are then loaded into a single lane or
multiple lanes, as desired. Next, an electrical current is applied
to the gel and extension products.
[0040] Subsequently, the temperature of the denaturing gel is
gradually raised, while maintaining the electrical current, so as
to separate the extension products on the basis of their melting
behaviors. Once the fragments have been separated by size and
melting behavior, one can identify the presence or absence of
mutations or polymorphisms at the screened loci by analyzing the
migration behavior of the extension products.
[0041] In other approaches, the extension products are isolated
from the reactants and suspended in a DHPLC buffer (e.g., 0.1M TEAA
pH 7.0). The extension products are then injected onto a DHPLC
column (e.g., an ion-pair reverse phase HPLC column composed of
alkylated non-porous poly(styrene-divinylbenzene)) that has been
equilibrated to an appropriate denaturing temperature, depending on
the size and composition of the DNA to be separated (e.g.,
53.degree. C. to 63.degree. C.) in an appropriate buffer (e.g., 0.1
mM triethylamine acetate (TEAA) pH 7.0) and the extension products
are allowed to bind. The presence of a denaturant (e.g.,
acetonitrile in TEAA) on the column is gradually raised over time
so as to slowly elute the extension products from the column.
Preferably a linear gradient is used. Presence of the extension
products in the eluant is preferably accomplished using a UV
detector (e.g., at 260 and/or 280 nm), however, greater sensitivity
may be obtained using radioactivity, binding dyes, fluorescence or
the techniques described in U.S. Pat. Nos. 5,795,976; 5,585,236;
6,024,878; 6,210,885; Huber, et al., Chromatographia 37:653 (1993);
Huber, et al., Anal. Biochem. 212:351 (1993); Huber, et al., Anal.
Chem. 67:578 (1995); and O'Donovan et al., Genomics 52:44 (1998),
which are all hereby incorporated by reference in their entireties
including the references cited therein.
[0042] The appearance of a slower or faster migrating band at a
temperature below or above the predicted melting point for the
particular extension product in the TTGE approach, for example,
indicates the presence of a mutation or polymorphism in the
subject's DNA. Similarly, the appearance of a slower or faster
eluting peak at a concentration of denaturant predicted to elute a
wild-type or non-polymorphic homoduplex extension product in the
DHPLC approach indicates the presence of a mutation or polymorphism
in the subject's DNA. A heterozygous sample will display both
homoduplex bands (wild-type homoduplexes and mutant homoduplexes),
as well as, two heteroduplex bands that are the product of
mutant/wild-type annealing. Because of base pair mismatches in
these fragments, they melt significantly sooner than the two
homoduplex bands. Accordingly, a user can rapidly identify the
presence or absence of a mutation or polymorphism at the screened
loci by either the TTGE or DHPLC approach and determine whether the
tested subject has a predilection for a disease.
[0043] In a related embodiment, greater sensitivity is obtained by
adding a "standard" DNA or "control" DNA to the DNA to be screened
prior to amplification or after amplification, prior to separation
of the DNA on the TTGE gel or DHPLC column. This insures the
presence of heteroduplexes in the case of either a homozygous
mutant, which normally would not display heteroduplexes, or a
heterozygous mutant. Desired DNA standards include, but are not
limited to, DNA that is wild-type for at least one of the traits
that are being screened. Preferred standards include, but are not
limited to, DNA that is wild-type for all of the traits that are
being screened. A DNA standard can also be a mutant or polymorphic
DNA. In some embodiments, particularly when the control DNA is
added after amplification, the DNA standard is an extension product
generated from a wild-type genomic DNA or a mutant genomic DNA. By
this approach, the amplification phase of the method is performed
as described above. That is, DNA from the subject to be screened
and the DNA standard are hybridized with nucleic acid primers that
flank regions of the genetic loci or markers that are associated
with or linked to the traits being tested.
[0044] Extension products are then generated. If the subject being
tested has at least one trait that is detected by the assay (e.g.,
a congenital disorder), then two populations of extension products
are generated, a first population that corresponds to the standard
DNA and a second population that corresponds to the subject's DNA
having at least one mutation or polymorphism. Next, preferably, the
two populations of extension products are isolated from the
amplification reactants and are denatured by heat (e.g., 95.degree.
C. for 5 minutes), then are allowed to anneal by cooling (e.g., ice
for 5 minutes). This ensures the formation of the heteroduplex
bands in the presence of any relatively small mutation (e.g., point
mutation, small insertion, or small deletion). The isolation and
denaturing/annealing steps are not practiced with some embodiments,
however.
[0045] Subsequently, by the TTGE approach, the two populations of
extension products are suspended in a non-denaturing loading buffer
and loaded on a denaturing polyacrylamide gel and separated on the
basis of melting behavior, as described above. By the DHPLC
approach, the two populations of extension products are suspended
in a suitable buffer (e.g., 0.1M TEAA pH 7.0), loaded onto a buffer
and temperature equilibrated DHPLC column and a linear gradient of
denaturant is applied, as described above. Because the two
populations of extension products are not perfectly complementary,
they form heteroduplexes. Heteroduplexes are less stable than
homoduplexes, have a lower melting temperature, and are easily
differentiated from homoduplexes using the DNA separation
techniques described above. One can identify the presence or
absence of mutations or polymorphisms at the screened loci, for
example, by comparing the migration behavior or elution behavior of
the extension products generated from the screened DNA with the
migration behavior or elution behavior of the DNA standard. If
heteroduplexes are present, generally, two additional bands that
correspond to the single extension product will appear on the gel
or the extension products will elute from the column more rapidly
than the control or standard DNA alerting the user to the presence
of a mutation or polymorphism. Accordingly, a significant increase
in sensitivity is obtained and a user can rapidly identify the
presence or absence of a mutation or polymorphism in the tested DNA
sample and, thereby, determine whether the screened subject has a
predilection for a particular trait (e.g., a congenital
disease).
[0046] Similarly, an increase in sensitivity can be obtained by
mixing DNA from a plurality of subjects prior to amplification.
Because the frequency of mutations or polymorphisms for most
disorders are very low in the population, most of the extension
products generated are wild-type DNA. Thus, most of the pool of DNA
behaves as a DNA standard. That is, the predominant structure
formed upon annealing after denaturation is a homoduplex, which can
be rapidly distinguished from any heteroduplex that would appear if
a subject were to have a polymorphism or mutation. Of course,
extension products previously generated from multiple subjects can
be used as control DNA by mixing the previously generated extension
products with the extension products generated from the DNA that is
being screened prior to electrophoresis. In several embodiments,
the DNA from at least 2 subjects is mixed. Desirably, the DNA from
at least 3 subjects is mixed. Preferably, the DNA from at least 4
subjects is mixed. It should be understood, however, that the DNA
from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, or more subjects can be mixed prior to
amplification or prior to separation on the basis of melting
behavior, in accordance with some of the described embodiments.
[0047] In one embodiment, for example, DNA from a plurality of
subjects to be tested is obtained by conventional methods, pooled,
and hybridized with the desired nucleic acid primers. Extension
products are then generated, as before. If at least one of the
subjects being tested has at least one congenital disorder that is
detected by the screen then two populations of extension products
will be generated, a first population that corresponds to DNA from
subjects that have the wild-type gene and a second population that
corresponds to DNA from subjects having at least one mutant or
polymorphic gene.
[0048] By one approach, the two populations of extension products
are then isolated from the amplification reactants, suspended in a
non-denaturing loading buffer, denatured by heat, annealed by
cooling, and are separated by TTGE, as described above. By another
approach, the two populations of extension products are isolated
from the amplification reactants, suspended in a DHPLC loading
buffer (0.1M TEAA pH 7.0), denatured by heat, annealed by cooling,
and are separated on a DHPLC column, as described above. The
presence of a subject in the DNA pool having at least one mutation
or polymorphism is identified by analyzing the migration behavior
of the DNA on the gel or the elution behavior from the column. The
appearance of a slower or faster migrating band at a temperature
below or above the predicted melting point for a particular
extension product on the gel indicates the presence of a mutation
or polymorphism in the DNA from one of the subjects. Similarly, the
appearance of a slower or faster eluting extension product from the
DHPLC column indicates the presence of a mutation or polymorphism
in the DNA from one of the subjects. By repeating the analysis with
smaller and smaller pools of samples, one can identify the
individual(s) in the pool that has the mutation or polymorphism.
Additionally, DNA standards can be used, as described above, to
facilitate identification of the individual(s) having the mutation
or polymorphism. Advantageously, some embodiments can be used to
screen multiple samples at multiple loci that are on found on a
plurality of genes in a single assay, thus, increasing sample
throughput. The analysis of a plurality of DNA samples in the same
assay also unexpectedly provides greater sensitivity. The section
below describes a DNA separation technique that can be used with
the embodiments described herein.
[0049] Multiple Extension Products of Similar Composition can be
Separated on the Same Lane of a Denaturing Gel or in the Same Run
on a DHPLC Column
[0050] It was discovered that multiple fragments of DNA, which vary
slightly in length and/or composition, can be rapidly and
efficiently resolved on the basis of melting behavior. Although the
preferred methods for differentiating multiple fragments of DNA on
the basis of melting behavior involve TTGE gel electrophoresis and
DHPLC, it is contemplated that other conventional techniques that
are amenable to DNA separation on the basis of melting behavior can
be equivalently employed (e.g., size exclusion chromatography, ion
exchange chromatography, and reverse phase chromatography on high
pressure (e.g., HPLC), low pressure (e.g., FPLC), gravity-flow, or
spin-columns, as well as, thin layer chromatography).
[0051] By one approach, a polyacrylamide gel having a porosity
sufficient to resolve the DNA fragments on the basis of size (e.g.,
4-20% acrylamide/bis acrylamide gel having a set concentration of
denaturant) is used. The amount of denaturant in the gel (e.g.,
urea or formamide) can vary according to the length and composition
of the DNA to be resolved. The concentration of urea in a
polyacrylamide gel, for example, can be 3M, 3.5M, 4M, 4.5M, 5M,
5.5M, 6M, 6.5M, 7M, 7.5M, or 8M. In preferred embodiments, an 8%
polyacrylamide gel with 7M urea is used. It should be emphasized,
however, that other types of polyacrylamide gels, equivalents
thereof, and agarose gels can be used.
[0052] The DNA samples to be resolved are placed in a
non-denaturing buffer and can be loaded directly to the gel. In
some embodiments, for example, when heteroduplex formation is
desired to increase the sensitivity of the assay, it is desirable
to heat the double stranded DNA to a temperature that permits
denaturation (e.g., 95.degree. C. for 5-10 minutes) and then slowly
cool the DNA to a temperature that allows annealing (e.g., ice for
5-10 minutes) prior to mixing with the loading buffer. Preferably,
the DNA is loaded onto the gel in a total volume of 10-20 .mu.l.
Preferably, a Temporal Temperature Gradient Gel Electrophoresis
(TTGE) apparatus is used. A commercially available system that is
suitable for this technique can be obtained from BioRad. The gel
can be run at 120, 130, 140, 150, 175, 200, 220, 250, 275, or 300 V
for 1.5-10 hours, for example.
[0053] Once the DNA has been loaded, an electrical current is
applied to begin separating the fragments on the bass of size and
the temperature of the gel is raised gradually. In one embodiment,
for example, the melting behavior separation is performed by
raising the temperature beyond 60.degree. C., 61.degree. C.,
62.degree. C., 63.degree. C., 64.degree. C., 65.degree. C.,
66.degree. C., 67.degree. C., 68.degree. C., 69.degree. C.,
70.degree. C., 71.degree. C., 72.degree. C., 73.degree. C.,
74.degree. C., or 75.degree. C. at approximately 5.0.degree.
C./hour-0.5.degree. C./hour in 0.1.degree. C. increments.
[0054] Once the extension products have been separated by melting
behavior, the gel can be stained to reveal the separated DNA. Many
conventional stains are suitable for this purpose including, but
not limited to, ethidium bromide stain (e.g., 1% ethidium bromide
in a 1.25.times.Tris Acetate EDTA pH 8.0 (TAE) solution),
fluorescent stains, silver stains, and colloidal gold stains. In
some embodiments, it is desirable to destain the gel (e.g., 20
minutes in a 1.25.times.TAE solution). After staining, the gel can
be analyzed visually (e.g., under a U.V. lamp) and/or with a
digital camera and computer software such as, the Eagle Eye System
by Stratagene or the Gel Documentation System (BioRad).
[0055] Mutations or polymorphisms are easily identified by
comparing the migration behavior of the DNA to be screened with the
migration behavior of a control DNA and/or by monitoring the
melting temperature of the extension products generated from the
screened DNA. Desirable "control" DNA or "standard" DNA includes a
DNA that is wild-type or non-polymorphic for at least one loci that
is screened and preferred standard DNA is wild-type or
non-polymorphic for all of the loci that are being screened.
Because this DNA separation technique is sufficiently sensitive to
identify a single base pair substitution in a DNA fragment up to
600 base pairs in length, small changes in the melting behaviors
and migration of the extension products can be rapidly
identified.
[0056] By another approach, DHPLC is used to resolve heteroduplex
and homoduplex molecules of several PCR extension products in a
single assay. Preferably, the heteroduplex and homoduplex extension
products are separated from each other by ion-pair reverse phase
high performance liquid chromatography. In one embodiment, a DHPLC
column that contains alkylated non-porous
poly(styrene-divinylbenzene) is used. Preferably, the DHPLC column
is equilibrated in an appropriate degassed buffer, referred to as
Buffer "A" (e.g., 0.1M TEAA pH 7.0) and is kept at a constant
temperature somewhat below the predicted melting temperature of the
extension products (e.g., 53.degree. C.-60.degree. C., preferably
50.degree. C.). A plurality of extension products that may be
generated from a plurality of different loci, as described herein,
are suspended in Buffer A and are injected onto the DHPLC column.
The Buffer A is then allowed to run through the column for a time
sufficient to insure that the extension products have adequately
bound to the column. Preferably, flow rate and the amount of gas
(e.g., argon or helium) are adjusted and kept constant so that the
pressure on the column does not exceed the recommended level.
Gradually, degassed denaturing buffer, referred to as Buffer "B",
(e.g., 0.1M TEAA pH 7.0 and 25% acetonitrile) is applied to the
column. Although an isocratic gradient can be used, a gradual
linear gradient is preferred. By one approach, to separate
fragments that range in size from 200-450 bp, for example, a
gradient of 50%-65% Buffer B (0.1M TEAA pH 7.0 and 25%
acetonitrile) is used. Of course, as the size of extension products
to be separated on the DHPLC column decreases, the gradient and/or
the amount of denaturant in Buffer B can be reduced, whereas, as
the size of extension products to be separated on the DHPLC column
increases, the gradient and/or the amount of denaturant in Buffer B
can be increased.
[0057] The DHPLC column is designed such that double stranded DNA
binds well but as the extension products become partially denatured
the affinity to the column is reduced until a point is reached at
which the particular extension product can no longer adhere to the
column matrix. Typically, heteroduplexes denature before
homoduplexes, thus, they would be expected to elute more rapidly
from the column than homoduplexes.
[0058] In some embodiments, particularly embodiments concerning the
separation of a plurality of different extension products (e.g.,
extension products generated from a plurality of loci), the choice
of primers and, thus, the extension products generated therefrom,
requires careful design. For example, a GC-clamp or other
artificial sequence can be used to adjust the melting
characteristics and increase the length of a particular DNA
fragment, if needed, to facilitate separation on the DHPLC or
improve resolution of the extension products. By one approach, each
set of primers in a multiplex reaction are designed and selected to
generate an extension product that has a unique homoduplex and
heteroduplex elution behavior. In this manner, each species can be
easily identified.
[0059] By another approach, each set of primers are designed to
generate extension products that have homoduplexes with very
similar melting characteristics. By this strategy, all of the
homoduplexes will elute at the same or very similar concentration
of denaturant, which is different than the concentration of
denaturant required to elute the heteroduplexes. Accordingly, the
elution of a species of extension product outside of the expected
range for the homoduplexes indicates the presence of a mutation or
polymorphism.
[0060] In the case that the extension products happen to have
overlapping retention times/elution behaviors, the DHPLC conditions
can be adjusted to include a primary separation on the basis of
size prior to increasing the concentration of the denaturant on the
column to improve resolution. The techniques described in Huber, et
al., Anal. Chem. 67:578 (1995), hereby expressly incorporated by
reference in its entirety, can be adapted for use with the novel
DHPLC separation approach described herein. In one embodiment, for
example, the alkylated non-porous poly(styrene-divinylbenzene)
DHPLC column can be used to separate the extension products on the
basis of size for a time sufficient to group the various
populations of extension products (i.e., the homoduplexes and
heteroduplexes generated from a single independent set of primers
constitute a single population of extension products) prior to
separating on the basis of melting behavior.
[0061] By one approach, the extension products are applied to the
column, as above, in Buffer A and a shallow linear gradient of
Buffer B (e.g., 30%-50% of a solution of 0.1M TEAA pH 7.0 and 25%
acetonitrile for 200-450 bp extension products) is applied so as to
resolve the various populations of extension products. Then, a
deeper linear gradient of Buffer B (e.g., 50%-65% of a solution of
0.1M TEAA pH 7.0 and 25% acetonitrile for 200-450 bp extension
products) is applied to resolve the homoduplexes from the
heteroduplexes within each individual population of extension
product. In this manner, the homoduplexes and heteroduplexes from
each population of extension product can be resolved despite having
overlapping elution behaviors.
[0062] It should be understood that the separation based on size
can be performed at virtually any temperature as long as the
extension products do not denature on the column, however, the
amount of denaturant in Buffer B and the type of gradient may have
to be adjusted. For example, the size separation can be
accomplished at 4.degree. C.-23.degree. C., or 23.degree.
C.-40.degree. C., or 40.degree.-50.degree. C., or 50.degree.
C.-60.degree. C. Additionally, the size separation can be
accomplished while the column is being gradually equilibrated to
the temperature that is going to be used for the DHPLC. It should
also be understood that the size separation can be performed on the
same column with the appropriate gradient (shallow for a time
sufficient to separate on the basis of size followed by a deeper
gradient to separate on the basis of melting behavior).
Additionally, columns in series can be used to separate extension
products that have overlapping retention times/elution behaviors.
For example, a first DHPLC column can be used to separate on the
basis of size and a second DHPLC column can be used to separate on
the basis melting behavior.
[0063] Mutations or polymorphisms are easily identified using the
DHPLC techniques above by comparing the elution behavior of the DNA
to be screened with the elution behavior of a control DNA. As
above, desirable "control" DNA or "standard" DNA includes a DNA
that is wild-type or non-polymorphic for at least one loci that is
screened and preferred standard DNA is wild-type or non-polymorphic
for all of the loci that are being screened. Control or standard
DNA can also include extension products that are homoduplexes by
virtue of a mutation or polymorphism or plurality of mutations or
polymorphisms. Since the elution behavior of the wild type or
non-polymorphic DNA or a homozygous mutant or polymorphism,
represents the elution behavior of a homoduplex, one can use DHPLC
values obtained from separating these controls, such as the
retention time, elution time, or amount of denaturant required to
elute the homoduplex as a basis for comparison to a screened sample
to identify the presence of homoduplexes. Similarly, a control DNA
can be a known heteroduplex and the elution behavior values
described above can be used to identify the presence of a
heteroduplex in a screened sample.
[0064] Additionally, the separated extension products can be
collected after passing through the DHPLC column or TTGE gel or
reamplified and sequenced to verify the existence of the mutation
or polymorphism. Further, the identified products can be isolated
from the gel and sequenced. Sequencing can be performed using the
conventional dideoxy approach (e.g., Sequenase kit) or an automated
sequencer. Preferably, all possible mutant fragments are sequenced
using the CEQ 2000 automated sequencer from Beckman/Coulter and the
accompanying analysis software. The mutations or polymorphisms
identified by sequencing can be compiled along with the respective
melting behaviors and the sizes of extension products. This data
can be recorded in a database so as to generate a profile for each
loci.
[0065] Additionally, this profile information can be recorded with
other subject-specific information, for example family or medical
history, so as to generate a subject profile. By creating such
databases, individual mutations can be better characterized.
Mutation analysis hardware and software can also be employed to aid
in the identification of mutations or polymorphisms. For example,
the "ALFexpress II DNA Analysis System", available from Amersham
Pharmacia Biotech and the "Mutation Analyser 1.01", also available
from Amersham Pharmacia Biotech, can be used. Mutation Analyser
automatically detects mutations in sample sequence data, generated
by the ALFexpress II DNA analysis instrument. The section below
describes embodiments that allow for the identification of a
mutation or polymorphism at multiple loci in a plurality of genes
in a single assay.
[0066] Identification of the Presence or Absence of a Mutation or
Polymorphism at Multiple Loci in a Plurality of Genes in a Single
Assay
[0067] The DNA separation techniques described herein can be used
to rapidly identify the presence or absence of a mutation or
polymorphism at multiple loci in a plurality of genes in a single
assay. Accordingly, a biological sample containing DNA is obtained
from a subject and the DNA is isolated by conventional means. For
some applications, it may be desired to screen the RNA of a subject
for the presence of a genetic disorder (e.g., a congenital disease
that arises through a splicing defect). In this case, a biological
sample containing RNA is obtained, the RNA is isolated, and then is
converted to cDNA by methods well known to those of skill in the
art. DNA from a subject or cDNA synthesized from the mRNA obtained
from a subject can be easily and efficiently isolated by various
techniques known in the art. Also known in the art is the ability
to amplify DNA fragments from whole cells, which can also be used
with the embodiments described herein. Thus, the DNA sample for use
with the embodiments described herein need only be isolated in the
sense that the DNA is in a form that allows for PCR
amplification.
[0068] In some embodiments, genomic DNA is isolated from a
biological sample by using the Amersham Pharmacia Biotech
"GenomicPrep Blood DNA Isolation Kit". The isolation procedure
involves four steps: (1) cell lysis (cells are lysed using an
anionic detergent in the presence of a DNA preservative, which
limits the activity of endogenous and exogenous Dnases); (2) RNAse
treatment (contaminating RNA is removed by treatment with RNase A);
(3) protein removal (cytoplasmic and nuclear proteins are removed
by salt precipitation); and (4) DNA precipitation (genomic DNA is
isolated by alcohol precipitation). EXAMPLE 1 also describes an
approach that was used to isolate DNA from human blood.
[0069] Once the sample DNA has been obtained, primers that flank
the desired loci to be screened are designed and manufactured.
Preferably, optimal primers and optimal primer concentrations are
used. Desirably, the concentrations of reagents, as well as, the
parameters of the thermal cycling are optimized by performing
routine amplifications using control templates. Primers can be made
by any conventional DNA synthesizer or are commercially available.
Optimal primers desirably reduce non-specific annealing during
amplification and also generate extension products that resolve
reproducibly on the basis of size or melting behavior and,
preferably, both. Preferably, the primers are designed to hybridize
to sample DNA at regions that flank loci that can be used to
diagnose a trait, such as a congenital disease (e.g., loci that
have mutations or polymorphisms that indicate a human disease).
[0070] Desirably, the primers are designed to detect loci that
diagnose conditions selected from the group consisting of familial
hypercholesterolemia (FH), cystic fibrosis, Tay-sachs, thalassemia,
sickle cell disease, phenylketonuria, galactosemia, fragile X
syndrome, hemophilia A, myotonic dystrophy, medium-chain acyl CoA
dehydrogenase, maturity onset diabetes, cystinuria, methylmolonic
acidemia, urea cycle disorders, hereditary fructose intolerance,
hereditary hemachromatosis, neonatal thrombocytopenia, Gaucher's
disease, tyrosinemia, Wilson's disease, alcaptonuria, hypolactasia,
Baker's disease, argininemia Adenomatous polyposis coli (APC),
Adult Polycystic Kidney disease, a-1-antitrypsin deficiency,
Duchenne Muscular Dystrophy, Hemophilia A, Hereditary Nonpolyposis
colorectal cancer, Huntingtons disease, Marfans syndrome, Myotonic
dystrophy, Neurofibromatosis, Osteogenesis imperfecta,
Retinoblastoma, Sickle cell disease, Freidrichs ataxia,
Hemoglobinopathies, Leber's hereditary optic neuropathy, MCAD,
Canavan's disease, Retintitus Pigmentosa, Bloom Syndrome, Fanconi
anemia, and Neimann Pick disease. Primers can be designed to
amplify any region of DNA, however, including those regions known
to be associated with diseases such as alcohol dependence, obesity,
and cancer. It should be understood that the embodiments described
herein can be used to detect any gene, mutation, or polymorphism
found in plants, virus, molds, yeast, bacteria, and animals.
[0071] Preferred primers are designed and manufactured to have a GC
rich "clamp" at one end of a primer, which allows the dsDNA to
denature in a "zipper-like" fashion. As one of skill will
appreciate, PCR requires a "primer set", which includes a first and
a second primer, only one of which has the GC clamp so as to allow
for separation of the double stranded molecule from one end only.
Since the GC clamp is significantly stable, the rest of the
fragment melts but does not completely separate until a point after
the inflection point of the DNA, which contains the mutation or
polymorphism of interest. The denaturant in the gel or on the
column allows the temperature of melting to be lower and allows the
inflection point of the melt to be longer in terms of temperature
and, thus, the sensitivity to temperature at the inflection point
is less (i.e., increment temperature=less increment melting), which
increases the resolution.
[0072] Additionally, desirable primers are designed with a properly
placed GC-clamp so that extension products that contain a single
melting domain are produced. Preferably, the primers are selected
to complement regions of introns that flank exons containing the
genetic markers of interest so that polymorphisms or mutations that
reside within the early portions of exons are not masked by the GC
clamp. For example, it was discovered that GC clamps significantly
perturb melting behavior and can prevent the detection of a
polymorphism or mutation by melting behavior if the mutation or
polymorphism resides too close to the GC clamp (e.g., within 40
nucleotides). By performing amplification reactions with control
templates, optimal primer design and optimal concentration can be
determined. The use of computer software, including, but not
limited to, WinMelt or MacMelt (Bio-Rad) and Primer Premire 5.0 can
aid in the creation and optimization of primers and proper
positioning of the GC-clamp. Accordingly, many of the primers and
groupings of primers described herein, as used in a particular
assay (e.g., to screen for cystic fibrosis) are embodiments of the
invention. EXAMPLE 2 further describes the design and optimization
of primers that allowed for the high-throughput multiplex PCR
technique described herein.
[0073] Once optimal primers are designed and selected, the DNA
sample is screened using the inventive multiplex PCR technique. In
some embodiments, for example, approximately 25 ng-500 ng of
template DNA (preferably, 200 ng for human genomic DNA) is
suspended in a buffer comprising: 10 mM Tris (pH 8.4), 50 mM KCl,
1.5 mM MgCl.sub.2, 200 .mu.M dNTPs, 50 pmol of each primer, and 1
unit Taq polymerase per primer set in a total volume of 50 .mu.l.
Preferably, amplification is performed under the same conditions
that were used to design the primers. In some embodiments, for
example, amplification is performed on a conventional thermal
cycler for 30 cycles, wherein each cycle is: 1 minute @ 95.degree.
C., 58.degree. C. for 1 minute, 72.degree. C. for 1 minute. Final
extension is performed at 72.degree. C. for 5 minutes. When the
primers have a GC clamp, it was found that conditions often favor
an amplification reaction having over 40 cycles, wherein each cycle
is: 35 seconds @ 95.degree. C., 120 seconds @ 50-57.degree. C., and
60 seconds+3 seconds/cycle @ 72.degree. C. Thermal cyclers are
available from a number of scientific suppliers and most are
suitable for the embodiments described herein.
[0074] Once the PCR reaction is complete, the extension products
are desirably isolated by centrifugal microfiltration using a
standard PCR cleanup cartridge, for example, Qiagen's QIAquick 96
PCR Purification Kit, according to manufacture's instructions.
Isolation or purification of the extension products is not
necessary to practice the invention, however. The isolated
extension products can then be suspended in a non-denaturing
loading buffer and either loaded directly on a DHPLC column or TTGE
denaturing gel. The sample can also be denatured by heating (e.g.,
95.degree. C. for 5-10 minutes) and annealed by cooling (e.g., ice
for 5-10 minutes) prior to loading onto the DHPLC column or TTGE
denaturing gel. The various extension products are then separated
on a TTGE denaturing gel or DHPLC column on the basis of melting
behavior, as described above and, after separation, the extension
products can be analyzed for the presence or absence of
polymorphisms or mutations. EXAMPLES 3 and 4 describe experiments
that verified that multiple loci on a plurality of genes can be
screened in a single assay. The section below describes a method of
genetic analysis, wherein improved sensitivity of detection was
obtained by adding a DNA standard to the screened DNA.
[0075] Improved Sensitivity was Obtained When a DNA Standard was
Mixed With the Screened DNA
[0076] It was also discovered that greater sensitivity in the
inventive multiplex PCR reactions described herein can be obtained
by mixing a DNA standard with the DNA to be tested prior to
conducting amplification or after amplification but prior to
separation on the basis of melting behavior. Desired DNA standards
include, but are not limited to, DNA that is wild-type for at least
one of the traits that are being screened and preferred DNA
standards include, but are not limited to, DNA that is wild-type
for all of the traits that are being screened. DNA standards can
also be mutant or polymorphic DNA. In some embodiments,
particularly when the control DNA is added after amplification, the
DNA standard is an extension product generated from a wild-type
genomic DNA or a mutant genomic DNA.
[0077] By one approach, the DNA from the subject to be screened and
the DNA standard are pooled and then the amplification reaction, as
described above, is performed. Accordingly, optimal primers are
designed and selected and approximately 25 ng-500 ng of template
DNA (preferably, 200 ng for human genomic DNA) is suspended in a
buffer comprising: 10 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM
MgCl.sub.2, 200 .mu.M dNTPs, 50 pmol of each primer, and 1 unit Taq
polymerase per primer set in a total volume of 50 .mu.l.
Preferably, amplification is performed under the same conditions
that were used to design the primers. In some embodiments,
amplification is performed on a conventional thermal cycler for 30
cycles, wherein each cycle is: 1 minute @ 95.degree. C., 58.degree.
C. for 1 minute, 72.degree. C. for 1 minute. Final extension is
performed at 72.degree. C. for 5 minutes. When the primers have a
GC clamp, however, conditions often favor an amplification reaction
having over 40 cycles, wherein each cycle is: 35 seconds @
95.degree. C., 120 seconds @ 50-57.degree. C., and 60 seconds+3
seconds/cycle @ 72.degree. C.
[0078] If the subject being tested has at least one disorder that
is detected by the assay then two populations of extension products
are generated, a first population that corresponds to the standard
DNA and a second population that corresponds to the subject's DNA
having at least one mutation or polymorphism. The pool of extension
products are desirably isolated from the amplification reactants,
as above, and are suspended in a non-denaturing loading buffer.
Preferably, the extension products are then denatured by heat
(e.g., 95.degree. C. for 5 minutes), and are allowed to anneal by
cooling (e.g., ice for 5 minutes) prior to loading on the TTGE
denaturing gel or DHPLC column. In this manner, the formation of
heteroduplexes will be favored if the subject has a mutation or
polymorphism because the two populations of extension products are
not perfectly complementary. However, the isolation and
denaturing/annealing steps are not necessary for some
embodiments.
[0079] By another approach, the DNA standard is added to the
extension products generated from the tested subject's DNA after
the amplification reaction. As above, the pooled DNA sample is
preferably denatured by heat (e.g., 95.degree. C. for 5 minutes),
and allowed to anneal by cooling (e.g., ice for 5 minutes). This
second approach also produces heteroduplexes if the extension
product and the DNA standard are not perfectly complementary.
[0080] Next, the TTGE denaturing gel or DHPLC column is loaded and
the extension products are separated on the basis of melting
behavior, as described above. Since heteroduplexes are less stable
than homoduplexes and have a lower melting temperature, the
presence or absence of a mutation or polymorphism in the tested DNA
sample is easily determined. By comparing the migration behavior or
elution behavior of the extension products generated from the
screened DNA with the migration behavior of the DNA standard, a
user can rapidly determine the presence or absence of a mutation or
polymorphism (e.g., two additional bands that correspond to the
single extension product will appear on the gel when a mutation or
polymorphism is present in the tested DNA or a population of
extension products will elute from the DHPLC column earlier than
homoduplex controls or the majority of homoduplexes present in the
sample). The section below describes a method of genetic analysis,
wherein improved efficiency and sensitivity of detection was
obtained by screening multiple DNA samples in the same assay.
[0081] Improved Sensitivity was Obtained When Multiple DNA Samples
Were Screened in the Same Assay
[0082] It was also discovered that an improved sensitivity of
detection and increased throughput could be obtained by mixing DNA
from a plurality of subjects prior to amplification. Because the
frequency of mutations or polymorphisms for most disorders are very
low in the population, most of the extension products generated
correspond to wild-type or non-polymorphic DNA. Accordingly, most
of the DNA in a reaction comprising DNA from a plurality of
subjects behave similar to a DNA standard. That is, the predominant
structure formed upon annealing after denaturation is a homoduplex,
which can be rapidly distinguished from any heteroduplex that would
appear if a subject were to have a mutation or polymorphism.
Although the reaction is "dirty" from the perspective that the
identity of each subject's DNA is not known initially, the identity
of any polymorphic or mutant DNA can be determined through a
process of elimination. For example, by repeating the analysis with
smaller and smaller pools of samples, one can identify the
individual(s) in the pool that have the mutation or polymorphism.
Additionally, DNA standards can be used, as described above, to
facilitate identification of the individual(s) having the mutation
or polymorphism.
[0083] By one approach, DNA from a plurality of subjects to be
tested is obtained by conventional methods, pooled, and hybridized
with the desired nucleic acid primers. Accordingly, optimal primers
are designed and selected and approximately 25 ng-500 ng of
template DNA (preferably, 200 ng for human genomic DNA) is
suspended in a buffer comprising: 10 mM Tris (pH 8.4), 50 mM KCl,
1.5 mM MgCl.sub.2, 200.mu.M dNTPs, 50 pmol of each primer, and 1
unit Taq polymerase per primer set in a total volume of 50 .mu.l.
Preferably, amplification is performed under the same conditions
that were used to design the primers. In some embodiments,
amplification is performed on a conventional thermal cycler for 30
cycles, wherein each cycle is: 1 minute @ 95.degree. C., 58.degree.
C. for 1 minute, 72.degree. C. for 1 minute. Final extension is
performed at 72.degree. C. for 5 minutes. When the primers have a
GC clamp, however, conditions often favor an amplification reaction
having over 40 cycles, wherein each cycle is: 35 seconds @
95.degree. C., 120 seconds @ 50-57.degree. C., and 60 seconds+3
seconds/cycle @ 72.degree. C.
[0084] The pool of extension products are preferably isolated from
the amplification reactants, as above, and are suspended in a
non-denaturing loading buffer. Preferably, the extension products
are then denatured by heat (e.g., 95.degree. C. for 5 minutes), and
are allowed to anneal by cooling (e.g., ice for 5 minutes). In this
manner, the formation of heteroduplexes will be favored if the
subject has a mutation or polymorphism because the two types of
extension products are not perfectly complementary. Again, the
isolation and denaturing/annealing steps are not performed in some
embodiments.
[0085] Next, the TTGE denaturing gel or DHPLC column is loaded and
the extension products are separated on the basis of melting
behavior, as described above. When one of the subjects being tested
has at least one trait that is detected by the screen,
heteroduplexes are detected on the gel or eluting from the DHPLC
column. The assay can be then repeated with smaller pools of
samples and assays with a DNA standard can be conducted with
individual samples to confirm the identity of the subject having
the mutation or polymorphism. EXAMPLE 5 describes an experiment
that verified that an improved sensitivity can be obtained by
mixing a plurality of DNA samples. EXAMPLE 6 describes an
experiment that verified that multiple genes and multiple loci
therein can be screened in a plurality of subjects, in a single
assay. EXAMPLE 7 describes the screening of multiple genes and
multiple loci therein, in a plurality of subjects, in a single
assay using a DHPLC approach. The section below describes the
optimization of primer design in the context of an approach that
was used to detect mutations and/or polymorphisms in the CFTR
gene.
[0086] Optimization of Primer Design and Extension Product Design
Facilitates Identification of Genetic Markers Associated With
Cystic Fibrosis
[0087] A preferred embodiment concerns the identification of the
presence or absence of genetic markers, mutations, or polymorphisms
in one or more subjects that are associated with cystic fibrosis.
By one approach, almost the entire CFTR gene was scanned for the
presence or absence of genetic markers, mutations, or polymorphisms
that contribute to cystic fibrosis. (See EXAMPLE 8). TABLE A
provides the sequences of exons of the CFTR gene and several
oligonucleotide primers that have been used to screen regions of
the CFTR gene for the presence or absence of genetic markers,
polymorphisms, and mutations that are associated with cystic
fibrosis. Where indicated, the notation (GC) refers to a GC clamp.
TABLE B also lists many oligonucleotide primers that have been used
to screen regions of the CFTR gene for the presence or absence of
genetic markers, polymorphisms, and mutations that are associated
with cystic fibrosis. TABLE B also shows starting and ending point
for each primer as it relates to the publicly available gene
sequence for the CFTR gene (GenBank Accession No. AH006034, the
contents of which are expressly incorporated by reference in its
entirety, also provided in SEQ. ID No. 45). It is contemplated that
primers that are any number between 5, 10, 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides
upstream or down stream of the primers identified in TABLE A or B
can be used with embodiments of the invention so long as these
primers produce extension products that melt over long stretches of
DNA (approximately 25, 50, 75, 100, 125, or 150 nucleotides) at
approximately the same temperature (within 0.degree. C.-1.5.degree.
C.) and are resolvable on a TTGE gel or DHPLC column. TABLE B
further provides the nucleotide positions on the CFTR gene (GenBank
Accession No. AH006034) that are 50 nucleotides upstream or down
stream of the listed oligonucleotides. In some embodiments, the
primers CF9T-s: (5' TAATGGATCATGGGCCATGT 3' (SEQ. ID. NO. 46)) and
CF9T-as: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGAAGAGACATG
GACACCAAAT 3' (SEQ. ID. NO. 47)) are also used.
[0088] The sequence of the CFTR gene sequence can also be obtained
from GenBank entries AC000061, or AC000111, all of which are herein
expressly incorporated by reference in their entireties.
Accordingly, embodiments include methods of diagnosing cystic
fibrosis with primers that are any number from 1-75 nucleotides
upstream or down stream from the beginning or ending of the primers
listed in TABLE A or B, preferably using the approaches described
herein. It is also preferred that said methods use primers that
produce extension products that melt over long stretches of DNA
(approximately 25, 50, 75, 100, 125, or 150 nucleotides) at
approximately the same temperature (within 0.degree. C.-1.5.degree.
C.) and are resolvable on a TTGE gel or DHPLC column. Preferably,
these extension products are obtained, grouped, and separated as
described below.
[0089] By one approach, samples of DNA were obtained from several
subjects to be screened using the approaches described herein and
were disposed in a plurality of 96-well micro-titer plates such
that a single row of each plate corresponded to a single tested
subject. In some cases, 7 total plates were used per assay, wherein
each plate has 7 sample lanes (i.e., 7 subjects analyzed) and an
eighth lane was used for positive control sample DNA. Amplification
buffer, amplification enzyme (e.g., Taq polymerase), and DNTPs were
added to the sample DNA in each well, as described above, and a
plurality of primer sets that encompass the most of the gene (e.g.,
61 primer sets) were to yield a final volume of 10 .mu.l. The
primer sets that were employed in a first set of tests are
identified in TABLE A. TABLE C describes the plate setup for these
amplification reactions, whereas TABLE D describes the conditions
for the TTGE separation for these tests, whereas TABLE E describes
the groupings for the various fragments for TTGE separation.
Preferred methods of diagnosing cystic fibrosis employ the primers
of TABLE A to generate extension products that are grouped
according to TABLE E and separated by melting behavior (e.g.,
TTGE). By using this approach, a rapid, inexpensive, and efficient
diagnosis of the presence or absence of a marker associated with
cystic fibrosis can be ascertained. The names of the extension
products, "fragments" in TABLE C, TABLE D, and TABLE E correspond
to the names of the primer sets used throughout. The "position"
refers to the location of the well on the 96 well plate and the
"Multi G" refers to the grouping pool of the extension products
prior to TTGE.
[0090] Although multiplex PCR reactions can be employed,
preferably, each primer set is run in an individual reaction.
Conditions for PCR were, in one case for example: 5 minutes at
96.degree. C. for initial denaturing followed by 35 total cycles
of: 30 seconds at 94.degree. C. and 30 seconds at the annealing
temperature or at a gradient of 49.degree. C. to 63.degree. C. and
a final 10 minutes at 72.degree. C. to complete synthesis of any
partial products. Most preferred are primers that have an annealing
temperature between 49.degree. C. and 63.degree. C., though many of
the primer sets have annealing temperatures that are at 49.degree.
C., 52.degree. C., 59.degree. C., and 62.4.degree. C. (See Appendix
H). An approximately 3.degree. C. window is allowed for each plate
(e.g., primers having annealing temperatures that are within
3.degree. C. of one another are grouped on a single plate).
Programs such as WINMELT were used to determine whether the primers
could be grouped into various primer sets that have similar
annealing temperatures so that individual groups of primers can be
amplified by Polymerase Chain Reaction (PCR) on the same plate.
[0091] Once the extension products had been generated they were
grouped, pooled, and mixed with loading dye. Thirteen Multi G
groups were used and the extension products "fragments" generated
by the various primer sets, which belong to one of the thirteen
groups are identified in TABLE C and TABLE E. After grouping and
pooling, the samples were loaded onto a TTGE gel. TABLE C also
lists the start and stop temperatures for the TTGE, for each Multi
G group. Preferably, the TTGE is run with a very shallow
temperature gradient, e.g., about 1.0.degree. C./hour for a total
of three hours, at high voltage, e.g., 150 volts. Once the
separation was complete, the gels were grouped, stained with
ethidum bromide, and analyzed by the Decode system. The analysis
above was rapid, inexpensive, and very effective at detecting
mutations and/or polymorphisms, many of which go undetected or are
not analyzed by others in the field.
[0092] Whereas many in the field seek to design primers that
optimally anneal with a template DNA, it has been discovered that
primers can also be designed to produce an optimal extension
product (e.g., a fragment of short length with a reliable and rapid
melting point). Preferably, primers are designed to generate
extension products that are approximately 100-300 nucleotides in
length and that have long stretches of DNA that melt at
approximately the same temperature (e.g., DNA stretches that are
25, 35, 45, 55, 65, 75, 85, 95, 100, 125, 15, 175, or 200
nucleotides that melt at the same temperature or within about a
0.degree. C. to about a 1.5.degree. C. temperature difference).
Programs such as WINMELT were used to evaluate the melting behavior
of extension products generated from the various primer sets and
test TTGE separation of the extension products generated by the
various primer sets were also performed to ensure that the
predicted melting behavior was represented on the gel. FIGS. 1-4
show graphs of four extension products produced by two of the
primer sets, described herein. The flat melting curve is preferred
for the applications described herein because the extension
products melt rapidly and are quickly retarded in the gel, which
improves resolution and allows multiple different extension
products to be separated in the same lane on a TTGE gel. That is,
by grouping extension products that have flat melting profiles,
which are within approximately 1.5.degree. C. of one another, it
allows a shallow TTGE temperature ramp (e.g., 1.degree. C. change
per hour for 3 hours) or shallow DHPLC temperature ramp, which
increases the sensitivity, allowing multiple extension products to
be separated in the same lane, which increases throughput and
reduces the cost of the analysis.
[0093] TABLE E shows several of the characteristics of the
extension products generated by the primers described herein. In
particular, the PCR annealing temperature for the primer set used
to generate the extension product ("PCR temp.") and a subjective
rating of performance is provided. The approximate melting
temperature ("App Tm") of the extension product and its length with
and without the GC clamp is provided. A range for the predicted
annealing temperature for the PCR and the range for the actual
annealing temperature for PCR is provided. The TTGE melting
temperature range is also given. Further, the Multi G group is also
listed. The following examples describe the foregoing methodologies
in greater detail. The first example describes an approach that was
used to isolate DNA from human blood.
EXAMPLE 1
[0094] A sample of blood was obtained from a subject to be tested
by phlebotomy. A portion of the sample (e.g., approximately 1.0 ml)
was added to approximately three times the sample volume or 3.0 ml
of a lysis solution (10 mM KHCO.sub.3, 155 mM NH.sub.4Cl, 0.1 mM
EDTA) and was mixed gently. The lysis solution and blood were
allowed to react for approximately five minutes. Next, the sample
was centrifuged (.times.500 g) for approximately 2 minutes and the
supernatant was removed. Some of the supernatant was left (e.g., on
the walls of the vessel) to facilitate suspension. The pellet was
then vortexed for approximately 5-10 seconds. An extraction
solution, which contains chaotropc and detergent (Qiagen), was then
added (e.g., 500 .mu.l), the sample was vortexed again for
approximately 5-10 seconds, and the solution was allowed to react
for five minutes at room temperature.
[0095] Next, a GFX column, which are pre-packed columns containing
a glass fiber matrix, was placed under vacuum (e.g., a Microplex 24
vacuum system) and the extracted solution containing the DNA was
transferred to the column (e.g., in 500 .mu.l aliquots). Once all
of the sample has been passed through the column, the vacuum was
allowed to continue for approximately 5 minutes. Subsequently, a
wash solution (Tris-EDTA buffer in 80% ethanol) was added (e.g.,
approximately 500 .mu.l) under vacuum. Once the wash solution had
been drained from the column, the vacuum was allowed to continue
for approximately 15 minutes. The GFX columns containing the DNA
were then placed into sterile microfuge tubes but the lids were
kept open.
[0096] Elution buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was then
added to the column (e.g., approximately 100 .mu.l of buffer that
was heated to approximately 70.degree. C.) and the buffer was
allowed to react with the column for approximately 2 minutes. Then,
the tubes containing the columns were centrifuged at .times.5000 g
for approximately 1.5 minutes. After centrifugation, the column was
discarded and the microfuge tube containing the isolated DNA was
stored at -20.degree. C. The example below describes the design and
optimization of primers that allowed for the inventive
high-throughput multiplex PCR technique, described herein.
EXAMPLE 2
[0097] Sets of primers for PCR amplification were designed for
every exon of the following genes: Cystic Fibrosis Transmembrane
Reductase (CFTR), Beta-hexosaminidase alpha chain (HEXA), PAH,
Alpha globin-2 (HBA2), Beta globin (HBB), Glucocerebrosidase (GBA),
Galactose-1-phosphae uridyl transferase (GALT), Medium chain
acyl-CoA dehydrogenase (MCAD), Protease inhibitor 1 (PI), Factor
VIII, FMR1, and Aspartoacylase (ASPA). The primers were designed
from sequence information that was available from GenBank or from
sequence information obtained from Ambry Genetics Corporation.
Information regarding mutations or polymorphisms was obtained from
The Human Gene Mutation Database.
[0098] One of the primers in each primer set contained a GC-clamp.
It was discovered that the addition of a GC-clamp significantly
altered the melting profile of the DNA extension product. Further,
proper positioning of the GC-clamp served to level the melting
profile. It was desired to position the GC-clamp so that a single
melting domain across the fragment was created. Proper positioning
of the GC-clamp was oftentimes needed to prevent the GC-clamp from
masking the presence of a mutation or polymorphism (e.g., if the
mutation or polymorphism is too close to the GC-clamp). Software
was also used to optimize primer design. For example, many primers
were designed with the aid of Primer Premiere 4.0 and 5.0 and
appropriate positioning of the GC-clamps was determined using
WinMelt software from BioRad. To maintain sensitivity of the test,
the primers were designed to anneal at a minimum of 40 base pairs
either upstream or downstream of the nearest known mutation in the
intronic region of the genes.
[0099] Although multiplex PCR can be technically difficult when
using the quantity of primers described herein, it was discovered
that almost all of the PCR artifacts disappeared when salt
concentration, temperature, primer selection, and primer
concentration were carefully optimized. Optimization was determined
for each primer set alone and in combination with other primer
sets. Optimization experiments were conducted using Master Mix from
Qiagen and a Thermocyler from MJ Research. The conditions for
thermal cycling were 5 minutes @ 95.degree. C. for the initial
denaturation, then 30 cycles of: 30 seconds @ 94.degree. C., 45
seconds @ 48-68.degree. C., and 1 minute @ 72.degree. C. A final
extension was performed at 72.degree. C. for 10 minutes.
[0100] In addition to primer compatibility, primers were selected
to facilitate identification of extension products by
electrophoresis. To optimize primer design in this regard, separate
PCR reactions were conducted for each individual set of primers and
the extension products were separated by the inventive DNA
separation technique, described above. Identical parameters were
maintained for each assay and the migration behavior for each
extension product was analyzed (e.g., compared to a standard to
determine a R.sub.f value for each fragment). An R.sub.f value is a
unit less value that characterizes a fragment's mobility relative
to a standard under set conditions. In many primer optimization
experiments, for example, the generated extension products were
compared to a standard extension product obtained from
amplification of the first exon of the PAH (phenylalanine
hydroxylase) gene. A measurement of the distance of migration of
each band in comparison to the distance of migration of the first
exon of PAH was recorded and the R.sub.f value was calculated
according to the following: 1 R f = ( migration distance of
fragment ) cm ( migration distance of PAH exon 1 ) cm
[0101] By conducting these experiments, it was verified that the
selected primers did not produce extension products that overlapped
on the gel. Optimal primer selection was obtained when optimal PCR
parameters were maintained and the extension products produced
dissimilar R.sub.f values. Finally, the multiplex PCR was tested
with all sets of primers and it was verified that few artifacts
were created during amplification. Embodiments of the invention
include the primers provided in the tables and sequence listing
provided herein and methods of using said primers and/or groups of
primers. The example below describes an experiment that verified
that the embodiments described herein effectively screen multiple
loci present on a plurality of genes in a single assay.
EXAMPLE 3
[0102] Two independent PCR reactions were conducted to demonstrate
that multiple loci on a plurality of genes can be screened in a
single assay using an embodiment of the invention. In a first
reaction, seven different loci from four different genes were
screened and, in the second reaction, eight different loci from
four different genes were screened. The primers used in each
multiplex reaction are provided in TABLE 1.
1 TABLE 1* Multiplex #1 Multiplex #2 Factor VIII 4 CFTR 23 (SEQ.
ID. Nos. 7 and 25) (SEQ. ID. Nos. 3 and 21) Factor VIII 11 CFTR 18
(SEQ. ID. Nos. 9 and 27) (SEQ. ID. Nos. 2 and 20) Factor VIII 24
Factor VIII 11 (SEQ. ID. Nos. 10 and 28) (SEQ. ID. Nos. 9 and 27)
PAH 9 Factor VIII 3 (SEQ. ID. Nos. 18 and 36) (SEQ. ID. Nos. 6 and
24) GBA 6 CFTR 24 (SEQ. ID. Nos. 15 and 33) (SEQ. ID. Nos. 37 and
38) Factor VIII 1 GBA 4 (SEQ. ID. Nos. 4 and 22) (SEQ. ID. Nos. 14
and 32) GALT 9 GALT 9 (SEQ. ID. Nos. 17 and 35) (SEQ. ID. Nos. 17
and 35) GBA 3 (SEQ. ID. Nos. 13 and 31) *Primers are stored in a 50
.mu.M storage stock and a 12.5 .mu.M working stock. Abbreviations
are: Phenyl alanine hydroxylase (PAH), Glucocerebrosidase (GBA),
Galactose-1-phosphate uridyl transferase (GALT), and cystic
fibrosis transmembrane reductase (CFTR). The numbers following the
abbreviations represent the exons probed.
[0103] The amplification was carried out in 25 .mu.l reactions
using a 2.times. Hot Start Master Mix, which contains Hot Start Taq
DNA Polymerase, and a final concentration of 1.5 mM MgCl.sub.2 and
200 .mu.M of each dNTP (commercially available from Qiagen). In
each reaction, 12.5 .mu.l of Hot Start Master Mix was mixed with 1
.mu.l of genomic DNA (approximately 200 ng genomic DNA), which was
purified from blood using a commercially available blood
purification kit (Pharmacia or Amersham). Primers were then added
to the mixture (0.5 .mu.M final concentration of each primer).
Then, ddH.sub.2O was added to bring the final volume to 25
.mu.l.
[0104] Thermal cycling for the Multiplex #1 reaction was performed
using the following parameters: 15 minutes @ 95.degree. C. for 1
cycle; 30 seconds @ 94.degree. C., 1 minute @ 53.degree. C., 1
minute and 30 seconds @ 72.degree. C. for 35 cycles; and 10 minutes
@ 72.degree. C. for 1 cycle. Thermal cycling for the Multiplex #2
reaction was performed using the following parameters: 15 minutes @
95.degree. C. for 1 cycle; 30 seconds @ 94.degree. C., 1 minute @
49.degree. C., 1 minute and 30 seconds @ 72.degree. C. for 35
cycles; and 10 minutes @ 72.degree. C. for 1 cycle.
[0105] After the amplification was finished, approximately 5 .mu.l
of each PCR product was mixed with 5 .mu.l of non-denaturing gel
loading dye (70% glycerol, 0.05% bromophenol blue, 0.05% xylene
cyanol, 2 mM EDTA). The DNA in the two reactions was then separated
on the basis of melting behavior on separate denaturing gels. Each
gel was a 16.times.16 cm, 1 mm thick, 7M urea, 8% acrylamidelbis
(37.5:1) gel composed in 1.25.times.TAE (50 mM Tris, 25 mM acetic
acid, 1.25 mM EDTA). Separation was conducted for 4 hours at 150 V
on the Dcode system (BioRad) and the temperature ranged from
51.degree. C. to 63.degree. C. with a temperature ramp rate of
3.degree. C./hour. Subsequently, the gels were stained in 1
.mu.g/ml ethidium bromide in 1.25.times.TAE for 3 minutes and
destained in 1.25.times.TAE buffer for 20 minutes. The gels were
then photographed using the Gel Doc 1000 system from BioRad.
[0106] The primers in TABLE 1 were selected and manufactured
because they produced extension products with very different
R.sub.f values and the extension products were clearly resolved by
separation on the basis of melting behavior. Although some bands
were more visible than others on the gel, seven distinct bands were
observed on the gel loaded with extension products generated from
the Multiplex 1 reaction and eight distinct bands were observed on
the gel loaded with extension products generated from the Multiplex
2 reaction. These results verified that the described method
effectively screened multiple loci on a plurality of genes in a
single assay. The example below describes another experiment that
verified that the embodiments described herein can be used to
effectively screen multiple loci present on a plurality of genes in
a single assay.
EXAMPLE 4
[0107] Experiments were conducted to differentiate extension
products generated from wild-type DNA and extension products
generated from mutant DNA. Samples of genomic DNA that had been
previously identified to contain mutations or polymorphisms were
purchased from Coriell Cell Repositories. The mutation or
polymorphism that was analyzed in this experiment was the
delta-F508 mutation of the CFTR gene. This mutation is a 3 bp
deletion in exon 10 of the CFTR gene. Other loci analyzed in these
experiments included the Fragile X gene, exon 17; Fragile X gene,
exon 3; Factor VIII gene exon 2; and the Factor VIII gene, exon 7.
Both the known mutant and a control wild-type for CFTR exon 10 were
amplified within a multiplex reaction and individually.
[0108] PCR amplification was conducted as described in EXAMPLE 3;
however, 0.25 .mu.M (final concentration) of each primer was used.
The primers used in these experiments were CFTR 10 (SEQ. ID. Nos. 1
and 19), FragX 17 (SEQ. ID. Nos. 12 and 30), FragX 3 (SEQ. ID.
Nos.11 and 29), Factor VIII 7 (SEQ. ID. Nos. 8 and 26) and Factor
VIII 2 (SEQ. ID. Nos. 5 and 23). The numbers following the
abbreviations represent the exons probed.
[0109] The DNA templates that were analyzed included known
wild-type genomic DNA, known mutant genomic DNA, mixed wild-type
genomic DNA from various subjects, and mixed wild-type and mutant
genomic DNA. Approximately 200 ng of genomic DNA was added to each
reaction. The mixed wild-type and mutant DNA sample had
approximately 100 ng of each DNA type. Thermal cycling was carried
out with a 15-minute. step at 95.degree. C. to activate the Hot
Start Polymerase, followed by 30 cycles of 30 seconds at @
94.degree. C., 1 minute at @ 53.degree. C., 1 minute and 30 seconds
at @ 72.degree. C.; and 72.degree. C. for 10 minutes.
[0110] After amplification, approximately 5 .mu.l of the PCR
product was mixed with 51 .mu.l of non-denaturing gel loading dye
(70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM
EDTA). The samples were then separated on a 16.times.16 cm, 1 mm
thick, 6M urea, 6% acrylamide/bis (37.5:1) gel in 1.25.times.TAE
(50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA) for 5 hours at 130 V
using the Dcode system (BioRad). The temperature ranged from
40.degree. C. to 50.degree. C. at a temperature ramp rate of
2.degree. C./hour. The gels were then stained in 1 .mu.g/ml
ethidium bromide in 1.25.times.TAE for 3 minutes and destained in
1.25.times.TAE buffer for 20 minutes. The gels were then
photographed using the Gel Doc 1000 system from BioRad.
[0111] The resulting gel revealed that the lane containing the
extension products generated from the wild-type DNA using the
CFTR10 primers had a mobility commensurate to the wild-type DNA
standard, as did the extension products generated from the other
primers and the wild-type DNA. That is, a single band appeared on
the gel in these lanes. The lane containing the extension products
generated from the template having the F508 mutant, on the other
hand, showed 2 bands. One of the bands had the same mobility as the
extension products generated from the wild-type or DNA standard and
the other band migrated slightly faster. These two populations of
bands represent the two populations of homoduplexes (i.e.,
wild-type/wild-type and mutant/mutant). The top band is the
wild-type homoduplex and the lower band is the mutant F508
homoduplex. Similarly, the lane that contained the wild-type/mutant
DNA mix exhibited two populations of extension products, one
representing the wild-type homoduplex population and the other
representing the mutant homoduplex. Since F508 is a 3 bp deletion
it failed to form heteroduplex bands in sufficient quantity to be
visible on the gel. Thus, this experiment demonstrated that the
described method effectively screened multiple genes, in a single
assay, and detected the presence of a polymorphism in one of the
screened genes. The example below describes an experiment that
demonstrated that an improved sensitivity can be obtained by mixing
a plurality of DNA samples.
EXAMPLE 5
[0112] This example describes two experiments that verified that an
improved sensitivity of detection can be obtained by (1) mixing the
DNA samples from a plurality of subjects prior to amplification or
by (2) mixing amplification products before separation on the basis
of melting behavior. In these experiments, PCR amplifications of
exon 9 of the GBA gene (Glucocerebrosidase gene) were used. DNA
samples known to contain a mutation in exon 9 of the GBA gene were
purchased from Coriell Cell Repositories. These DNA samples contain
a homozygous mutation in exon 9 of the GBA gene (the N370S
mutation).
[0113] In a first experiment, single amplification of exon 9 was
performed in a 25 .mu.l reaction. A Taq PCR Master Mix (containing
Taq DNA Polymerase and a final concentration of 1.5 mM MgCl.sub.2
and 200 .mu.M dNTPs)(Qiagen) was mixed with 0.5 .mu.M (final
concentration) of primers (SEQ. ID. Nos. 16 and 34). The template
genomic DNAs analyzed in this experiment included wild-type DNA,
mutant DNA, and various mixtures of wild-type and mutant DNA. For
the single non-mixed reactions, approximately 200 ng of genomic DNA
was used for amplification. In the mixed samples, approximately 200
ng of DNA was again used, however, the percentage of wild-type to
mutant genomic DNA varied. Thermal cycling was performed according
to the following parameters: 10 minutes @ 94.degree. C.; 30 cycles
of 30 seconds @ 94.degree. C., 1 minute @ 44.5.degree. C., and 1
minutes and 30 seconds @ 72.degree. C.; and 10 minutes @ 72.degree.
C.
[0114] In the second experiment, the amplification products were
mixed prior to separation on the basis of melting behavior.
Amplification of both wild-type and mutant (N370S) exon 9 of the
GBA gene was performed using 25 .mu.l reactions, as before. The Taq
Master Mix obtained from Qiagen was mixed with 200 ng of genomic
DNA and 0.5 .mu.M final concentration of both primers (SEQ. ID.
Nos. 16-34). PCR was carried out for 30 cycles with an annealing
temperature of 56.degree. C. for 1 minute. The denaturation and
elongation steps were 94.degree. C. for 30 seconds and 72.degree.
C. for 1 minute and 30 seconds. Final elongation was carried out at
72.degree. C. for 10 minutes. The extension products obtained from
the single amplification of wild-type GBA exon 9 was then mixed
with the extension products obtained from the single amplification
of the mutant GBA exon 9. Next, the pooled DNA was subjected to
denaturation at 95.degree. C. for 10 minutes and cooled on ice for
5 minutes, then heated to 65.degree. C. for 5 minutes and cooled to
4.degree. C. This denaturation and annealing procedure was
performed to facilitate the formation of heteroduplex DNA.
[0115] Once the extension products from both experiments were in
hand, approximately 5 .mu.l of both the prior to PCR mixture and
post PCR mixture were loaded on 16.times.16 cm, 1 mm thick gels (7M
Urea/8% acrylamide (37.5:1) gel in 1.25.times.TAE) using the gel
loading dye and the Dcode system (BioRad), described above. The DNA
on the gel was then separated at 150 V for 5 hours and the
temperature was uniformly raised 2.degree. C./hour throughout the
run starting at 50.degree. C. and ending at 60.degree. C. The gel
was stained in 1 .mu.g/ml ethidium bromide in 1.25.times.TAE buffer
for 3 minutes and destained in buffer for 20 minutes.
[0116] It should be noted that the GBA gene has a pseudo gene,
which was co-amplified by the procedure above. An extension product
generated from this psuedo gene migrated slightly faster than the
extension product generated from the true expressed gene on the
gel. In all lanes, the band representing the extension product
generated from the psuedo gene was present. Then next fastest band
on the gel was the extension product generated from the GBA exon 9
wild-type allele. The extension product generated from the mutant
GBA exon 9 allele comigrated with the wild-type allele and was
virtually indistinguishable on the basis of melting behavior due to
the single base difference.
[0117] The heteroduplexes formed in the mixed samples were easily
differentiated from the homoduplexes. The samples mixed prior to
PCR showed both homoduplexes (wild-type and mutant) along with
heteroduplexes, which appeared higher on the gel. Thus, by mixing
samples, either prior to amplification or prior to separation on
the basis of melting behavior an improved sensitivity of detection
was obtained. Since homoduplex bands no longer need to be resolved
to identify a mutation or polymorphism, only the heteroduplex bands
need to be resolved, the throughput of diagnostic analysis was
greatly improved. The example below describes experiments that
verified that the embodiments taught herein can be used to
effectively screen multiple genes in a plurality of subjects, in a
single assay, for the presence or absence of a polymorphism or
mutation.
EXAMPLE 6
[0118] Two experiments were conducted to verify that multiple genes
from a plurality of subjects can be screened in a single assay for
the presence or absence of a genetic marker (e.g. a polymorphism or
mutation) that is indicative of disease. These experiments also
demonstrated that an improved sensitivity of detection could be
obtained by mixing DNA samples either prior to generation of
extension products or prior to separation on the basis of melting
behavior.
[0119] In both experiments, five different extension products were
generated from three different genes in a single reaction vessel.
The five different extension products were generated using the
following primers: Factor VIII 1 (SEQ. ID. Nos. 4 and 22); GBA 9
(SEQ. ID. Nos. 16 and 34); GBA 11 (SEQ. ID. Nos. 39 and 40); GALT 5
(SEQ. ID. Nos. 41 and 42), and GALT 8 (SEQ. ID. Nos. 43 and 44).
Abbreviations are: Glucocerebrosidase (GBA) and
Galactose-1-phosphate uridyl transferase (GALT). The numbers
following the abbreviations represent the exons probed.
[0120] Extension products were generated for each experiment in
25.mu.l amplification reactions using Qiagen's 2.times. Hot Start
Master Mix (Contains Hot Start Taq DNA Polymerase, and a final
concentration of 1.5 mM MgCl.sub.2 and 200 .mu.M of each dNTP). To
each reaction, 12.5 .mu.l of Hot Start Master Mix was added to 1
.mu.l of genomic DNA (approximately 200 ng genomic DNA for the
mutant DNA sample and the wild-type DNA sample), which was purified
from human blood using Pharmacia Amersham Blood purification kits.
For the experiment in which the DNA samples from a plurality of
subjects were mixed prior to generation of the extension products,
approximately 100 ng of wild-type genomic DNA was mixed with
approximately 100 ng of mutant N370S genomic DNA. In both
experiments, primers were added to achieve a final concentration of
0.5 .mu.M for each primer and a final volume of 25 .mu.l was
obtained by adjusting the volume with ddH.sub.2O.
[0121] Thermal cycling for both experiments was performed using the
following parameters: 15 minutes @ 95.degree. C. for 1 cycle; 30
seconds @ 94.degree. C., one minute @ 57.degree. C., and one minute
30 seconds @ 72.degree. C. for 35 cycles; and 10 minutes @
72.degree. C. for 1 cycle. After amplification, the extension
products generated from the wild-type and mutant templates (the
un-mixed samples) were separated from the PCR reactants using a PCR
Clean Up kit (Qaigen). Then, approximately 10 .mu.L of the
wild-type and mutant DNA were removed from each tube and gently
mixed in a single reaction vessel. This preparation was then
denatured at 95.degree. C. for 1 minute and rapidly cooled to
4.degree. C. for 5 minutes. Finally, the preparation was brought to
65.degree. C. for an additional 1.5 minutes. The extension products
generated from the mixed sample (wild-type DNA and mutant DNA mixed
prior to amplification) were stored until loaded onto a denaturing
gel.
[0122] Next, approximately 10 .mu.l of the unmixed sample was
combined with 10 .mu.l of loading dye and approximately 5 .mu.l of
the mixed sample was combined with 5 .mu.l of loading dye. The
loading dye was composed of 70% glycerol, 0.05% bromophenol blue,
0.05% xylene cyanol, and 2 mM EDTA). The samples in loading dye
were then loaded on separate 16.times.16 cm, 1 mm thick, 7M urea,
8% acrylamidelbis (37.5:1) gels in 1.25.times.TAE (50 mM Tris, 25
mM acetic acid, 1.25 mM EDTA). The DNA was separated on the basis
of melting behavior for 5 hours at 150 V on the Dcode system
(BioRad). The temperature ranged from 56.degree. C. to 68.degree.
C. at a temperature ramp rate of 2.degree. C./hr. The gels were
then stained in 1 .mu.g/ml ethidium bromide in 1.25.times.TAE for 3
minutes and destained in 1.25.times.TAE buffer for 20 minutes. The
gels were photographed using the Gel Doc 1000 system (BioRad).
[0123] In all lanes of the gel, 5 extension products generated from
three different genes were visible in the following order from top
to bottom: Factor VIII 1, GBA 9, GBA 11, GALT 8, and GALT 5. Two
different extension products were generated from the GBA 9 primers,
as described above. The less intense band below the homoduplex
bands corresponded to an extension product generated from the
pseudogene. In the lanes loaded with extension products generated
from only the wild-type or mutant DNA template, it was difficult to
distinguish the wild type homoduplex from the N370S mutant
homoduplex. In the lane loaded with the extension products
generated from the mixed DNA templates (wild-type and mutant DNA
mixed prior to amplification) and the lane loaded with extension
products (generated from wild type and mutant DNA separately) that
were mixed after amplification, heteroduplex bands were easily
visualized. These experiments verified that multiple genes can be
screened in a plurality of individuals in a single assay and that a
single nucleotide mutation or polymorphism can be detected.
Further, these experiments demonstrate that screening a plurality
of DNA samples in a single reaction vessel or adding a control DNA
before or after amplification greatly improves the sensitivity of
detection. By practicing the methods taught in this example, the
throughput of diagnostic screening can be drastically improved and
the cost of identifying genetic traits can be significantly
reduced. The example below describes approaches to screen multiple
genes in a plurality of subjects, in a single assay, for the
presence or absence of a polymorphism or mutation using DRPLC.
EXAMPLE 7
[0124] Multiple genes in a plurality of subjects, in a single
assay, can be screened for the presence or absence of a
polymorphism or mutation using a DHPLC separation approach. For
example, five different extension products can be generated using
the following primers: Factor VIII 1 (SEQ. ID. Nos. 4 and 22); GBA
9 (SEQ. ID. Nos. 16 and 34); GBA 11 (SEQ. ID. Nos. 39 and 40); GALT
5 (SEQ. ID. Nos. 41 and 42), and GALT 8 (SEQ. ID. Nos. 43 and 44).
Abbreviations are: Glucocerebrosidase (GBA) and
Galactose-1-phosphate uridyl transferase (GALT). The numbers
following the abbreviations represent the exons probed. The
extension products can be generated in 25 .mu.l amplification
reactions using Qiagen's 2.times. Hot Start Master Mix (Contains
Hot Start Taq DNA Polymerase, and a final concentration of 1.5 mM
MgCl.sub.2 and 200 .mu.M of each dNTP).
[0125] To each reaction, 12.5 .mu.l of Hot Start Master Mix is
added to 1 .mu.l of genomic DNA (approximately 200 ng genomic DNA
for the mutant DNA sample and the wild-type DNA sample), which is
purified from human blood using Pharmacia Amersham Blood
purification kits. By another approach, the DNA samples from a
plurality of subjects can be mixed prior to generation of the
extension products. In this case, approximately 100 ng of wild-type
genomic DNA is mixed with approximately 100 ng of mutant N370S
genomic DNA. Primers are added to achieve a final concentration of
0.5 .mu.M for each primer and a final volume of 25 .mu.l is
obtained by adjusting the volume with ddH.sub.2O.
[0126] Thermal cycling is performed using the following parameters:
15 minutes @ 95.degree. C. for 1 cycle; 30 seconds @ 94.degree. C.,
one minute @ 57.degree. C., and one minute 30 seconds @ 72.degree.
C. for 35 cycles; and 10 minutes @ 72.degree. C. for 1 cycle. After
amplification, the extension products generated from the wild-type
and mutant templates (if un-mixed samples) are separated from the
PCR reactants using a PCR Clean Up kit (Qiagen). Then,
approximately 10 .mu.L of the wild-type and mutant DNA are removed
from each tube and gently mixed in a single reaction vessel. This
preparation is then denatured at 95.degree. C. for 1 minute and
rapidly cooled to 4.degree. C. for 5 minutes. Finally, the
preparation is brought to 65 .degree. C. for an additional 1.5
minutes. The extension products generated from the mixed sample
(wild-type DNA and mutant DNA mixed prior to amplification) can be
stored until loaded onto a DHPLC column.
[0127] Next, the extension products are loaded on to a 50.times.4.6
mm ion pair reverse phase HPLC column that is equilibrated in
degassed Buffer A (0.1 M triethylamine acetate (TEAA) pH 7.0) at
56.degree. C. A linear gradient of 40%-50% of degassed Buffer B
(0.1 M triethylamine acetate (TEAA) pH 7.0 and 25% acetonitrile) is
then performed over 2.5 minutes at a flow rate of 0.9 ml/min at
56.degree. C., followed by a linear gradient of 50%-55.3% Buffer B
over 0.5 minutes, and finally a linear gradient of 55.3%-61% Buffer
B over 4 minutes. U.V. absorption is monitored at 260 nm, recorded
and plotted against retention time.
[0128] When the loaded sample is un-mixed extension products, the
extension products generated from only the wild-type or mutant DNA
template, it is difficult to distinguish the wild type homoduplex
from the N370S mutant homoduplex. When the loaded sample is the
mixed extension products, the extension products generated from the
mixed DNA templates (wild-type and mutant DNA mixed prior to
amplification), or the extension products (generated from wild type
and mutant DNA separately) that were mixed after amplification,
heteroduplex elution behavior is detected. By practicing the
methods taught in this example, the throughput of diagnostic
screening can be drastically improved and the cost of identifying
genetic traits can be significantly reduced. The example below
describes an approach that was used to diagnostically screen
patient samples for cystic fibrosis.
EXAMPLE 8
[0129] Sets of primers for PCR amplification were designed for
every exon and one deep intronic region of the CFTR gene. The
primers were designed from sequence information that was available
from GenBank or from sequence information obtained from Ambry
Genetics Corporation. Information regarding mutations or
polymorphisms was obtained from The Human Gene Mutation
Database.
[0130] Primer sets and PCR stacking groups were designed for
optimal sensitivity for TTGE, as described above. DNA from one
individual was amplified with each primer set in a separate
reaction, then stacked in average groups of three fragments/gel for
gel analysis. Preferably, one of the primers in each primer set
contained a GC-clamp. It was discovered that the addition of a
GC-clamp significantly altered the melting profile of the DNA
extension product. Further, proper positioning of the GC-clamp
served to level the melting profile. It was desired to position the
GC-clamp so that a tight single melting domain across the fragment
was created. Proper positioning of the GC-clamp was often times
needed to prevent the GC-clamp from masking the presence of a
mutation or polymorphism (e.g., if the mutation or polymorphism is
too close to the GC-clamp). Software was also used to optimize
primer design. For example, many primers were designed with the aid
of Primer Premiere 4.0 and 5.0 and appropriate positioning of the
GC-clamps was determined using WinMelt software from BioRad. To
maintain sensitivity of the test, the primers were designed to
anneal at a minimum of 40 base pairs either upstream or downstream
of the nearest known mutation in the intronic region of the
genes.
[0131] Optimization was determined for each primer set.
Optimization experiments were conducted using Hotstart Master Mix
from Qiagen and a Thermocyler from MJ Research. Resulting PCR
conditions for all fragments were 15 minutes @ 95.degree. C. for
the initial denaturation, then 35 cycles of: 30 seconds @
94.degree. C., 30 seconds @ 46-62.degree. C., and 30 seconds @
72.degree. C. A final extension was performed at 72.degree. C. for
10 minutes. Approximately 15 .mu.l PCR reactions contained 7.5
.mu.l Qiagen 2.times. Hotstart Master Mix, 50-200 ng genomic DNA,
sense and antisense primer for each fragment at a final
concentration of 0.5-1 .mu.M. Prior to gel loading and stacking of
gel groups PCR samples were heated and re-annealed to provide best
heteroduplex formation. PCR product was heated to 95.degree. C. for
5 min, 50.degree. C. for 10 min, then brought to 4.degree. C.
[0132] On occasion, diagnostic patient samples may contain
mutations that are homozygous in nature, and sporadically
homozygous mutation band may settle in line with the wild-type
band. The most common mutation for CF (allele frequency .about.70%
known as dF508) has this situation. Therefore, wild-type gDNA was
always mixed with the diagnostic sample for exon 10 (primer set
10C) and heteroduplex formation was performed. This creates
heteroduplex bands which will predict a dF508, either homozygous or
heterozygous for the patient. Approximately 4 .mu.l of the
10C-amplified PCR sample from each patient was removed from the PCR
plate, transferred into 200 .mu.l strip tubes, mixed with 4 .mu.l
of 10C amplified wild type DNA, heated to 95.degree. C. for 5 min,
50.degree. C. for 10 min, 4.degree. C. and added back to the
assay.
[0133] PCR products (approximately 4-8 .mu.l each depending on
signal strength) were then assembled for groups of equal melting
characteristics and mixed with loading dye consisting of 70%
glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM EDTA).
DNA was separated on denaturing gels (7 M urea, 8% acrylamide/bis
(37.5:1) in 50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA) for 3-5
hours at 125 V or 150 V on the Dcode system. (Biorad). Temperature
ranged from 45.5.degree. C. to 64.degree. C. with ramp rates of
1.0-1.5.degree. C./hr depending on gel groups. The gels were
stained in 1 .mu.g/ml ethidium bromide in 1.25.times.TAE for 3
minutes and destained in 1.25.times.TAE buffer for 20 minutes. The
gels were photographed using the Gel Doc 1000 system (BioRad).
TABLE 2 lists the primers used in this assay. TABLE 3 shows the
TTGE gel grouping and temperatures used for TTGE separation. TABLE
3 also names the extension products generated from the various
primer sets employed and the positions of each fragment on the gel
after separation. Previous experiments, described above, have
demonstrated that extension products generated from primers that
are any number between 1-75 nucleotides upstream or downstream from
the primers listed in TABLE A (e.g., the primer sets listed in
TABLE 2) can be grouped and efficiently separated in accordance
with rules set forth herein. Preferably, the primers listed in
TABLE 2 are used to generate extension products that are grouped
according to TABLE 3 and are separated on the basis of melting
behavior (e.g., TTGE).
2TABLE 2 CFTR1A-s CGCCCGCCGCGCCCCGCGCGCCCGCCCCGCCGC-
CCCCGCCCGGGAAGCCAAATGA CATCACAGC (SEQ. ID. No. 48) CFTR1A-as
TGAAAAAAAGTTTGGAGACAACGC (SEQ. ID. No. 49) CFTR1B-s
CCCAGCGCCCAGAGACC (SEQ. ID. No. 50)
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGACTGCTTATTCCTTTA CFTR1B-as
CCCCAA (SEQ. ID. No. 51) CFTR2A-as2
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCCAGAAAAGTTGAAT AGTATCAG
(SEQ. ID. No. 52) CFTR2A-as2 AGATTGTCAGCAGAATCAA (SEQ. ID. No. 53)
CF2B-s5 ATACCAAATCCCTTCTG (SEQ. ID. No. 54) CF2B-as5
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGCTTTCTCTTCTCTA AAT (SEQ.
ID. No. 55) CF3A-s2 CGCCCGCCGCGCCCCGCGCCCGCCC-
CGCCGCCCCCGCCCGTGGTGTTGTATGGTCT (SEQ. ID. No. 56) CF3A-as2
AACATAAATCTCCAGAA (SEQ. ID. No. 57) CFTR3B-s GCTGGCTTCAAAGAAAAATCC
(SEQ. ID. No. 58) CFTR3B-as
CGCCCGCCGGGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCACCAGATTTCGTAGTCT TTTCA
(SEQ. ID. No. 59) CFTR4A-s CGCCCGCCGCGCCCCGCGCCCGCCCCGCCG-
CCCCCGCCCGAATTTCTCTGTTTTTCCCCTT (SEQ. ID. No. 60) CFTR4A-as
AGCTATTCTCATCTGCATTCCA (SEQ. ID. No. 61) CFTR4B-s GACACTGCTCCTACACC
(SEQ. ID. No. 62) CFTR4A-s
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTCAGCATTTATCCCTTA (SEQ. ID.
No. 63) CFTR5A-s CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCG-
CCCGATAATATATTTGTATTTTGT TTGTTG (SEQ. ID. No. 64) CFTR5A-as
AATTTGTTCAGGTTGTTGGA (SEQ. ID. No. 65) CFTR5B-s AGCTGTCAAGCCGTGTTC
(SEQ. ID. No. 66) CFTR5B-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATCTGACCCAGGAAAACTC (SEQ.
ID. No. 67) CFTR6A-1-s CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCC-
CGCCCGTTGTTAGTTTCTAGGGGTGG (SEQ. ID. No. 68) CFTR6A-1-as
AAGGACTATCAGGAAACCAAG (SEQ. ID. No. 69) CFTR6A-2-s
GCTAATCTGGGAGTTGTTAC (SEQ. ID. No. 70) CFTR6A-2-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGAGTTATGAAAATAGGTTGC AC
(SEQ. ID. No. 71) CF6A-3-s2
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGGGAGAATGATGATGAAG (SEQ.
ID. No. 72) CF6A-3-as2 ACACTGAAGATCACTGTTCTA (SEQ. ID. No. 73)
CFTR6B-1-s2 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGC-
CCGCCTTGAGCAGTTCTTAATAG ATA (SEQ. ID. No. 74) CFTR6B-1-as2
ATGCCTTAACAGATTGGATAT (SEQ. ID. No. 75) CFTR6B-2-s2
GAAAATATCCAATCTGTTAAG (SEQ. ID. No. 76) CFTR6B-2-as2
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGAGGTGGAAGTCTACCA (SEQ.
ID. No. 77) CFTR7A-s CGCCCGCCGCGCCCCGCGCCCGCC-
CCGCCGCCCCCGCCCGAGACCATGCTCAGATCTTC CATT (SEQ. ID. No. 78)
CFTR7A-as GCTGCCTTCCGAGTCAGTTTCAGT (SEQ. ID. No. 79) CFTR7C-s
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGACTGAAACTGACTCGG AAGG (SEQ.
ID. No. 80) CFTR7C-as ATGGTACATTACCTGTATTTTGTTTA (SEQ. ID. No. 81)
CFTR7D-s CTGTACAAAGATGGTATGACTCTCTT (SEQ. ID. No. 82) CFTR7D-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGTGAAGGAAATTTCTTTT TCTATCT
(SEQ. ID. No. 83) CFTR8A-s
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGCAGAATGAGAGTATAA AGTAG
(SEQ. ID. No. 84) CFTR8A-as CCATCACTACTTCTGTAGTCG (SEQ. ID. No. 85)
CF8B-s2: CTCTCTTTTATAAATAGGATTTCTTAC (SEQ. ID. No. 86) CF8B-as2:
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCC- GCCCGTTCCAGTTCTACCAGTT
ATATCATC (SEQ. ID. No. 87) CFTR9C-s
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGACAATAGAAAAACTTCTA ATGGTGA
(SEQ. ID. No. 88) CFTR9C-as AAAAAAGAGACATGGACACCAA (SEQ. ID. No.
89) CFTR10-s CCTGAGCGTGATTTGATA (SEQ. ID. No. 90) CFTR10-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATGTAGACTAACCGA TTGAA (SEQ.
ID. No. 91) CF10C-s3 GGGAGAACTGGAGCCT (SEQ. ID. No. 92) CF10C-as3
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGAAC- CGATTGAATA TGGAG (SEQ.
ID. No. 93) CFTR11A-s2
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGATATATGATTAC ATTAGAAG
(SEQ. ID. No. 94) CFTR11A-as2 ACCTTCTCCAAGAACTA (SEQ. ID. No. 95)
CFTR11B-s ATAGGACATCTCCAAGTT (SEQ. ID. No. 96) CFTR11B-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGCAATAGAG- AAA TGTCTGT
(SEQ. ID. No. 97) CFTR12-s
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGTGAACTGTTTAA GGCAAATCAT
(SEQ. ID. No. 98) CFTR12-as TGATGGGACAGTCTGTCTTTC (SEQ. ID. No. 99)
CFTR13A-s AATACGAGACATATTGCAATAAAGT (SEQ. ID. No. 100) CFTR13A-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGC- CCGCTGGCTGTAGATTT TGGAGTTC
(SEQ. ID. No. 101) CF13B-s3 AGGTAGCAGCTATTTTT (SEQ. ID. No. 102)
CF13B-as3 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGGACAGCCT
TCTCTCTA (SEQ. ID. No. 103) CFTR13C-s
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCC- CGCCCGATGGGACATTT TCAGAACTCC
(SEQ. ID. No. 104) CFTR13C-as CCTCTTCGATGCCATTCAT (SEQ. ID. No.
105) CFTR13E-s CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGATGAGCCT
TTAGAGAGAA (SEQ. ID. No. 106) CFTR13E-as CCAGTTCAGTCAAGTTTGC (SEQ.
ID. No. 107) CF13F-s2
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCAGCGTGA TCAGCA (SEQ. ID.
No. 108) CF13F-as2 TTTGTTTACATGCTACATA (SEQ. ID. No. 109)
CFTR14A-1-s CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTTCAT- ATATTA
AAAATAAAACC (SEQ. ID. No. 110) CFTR14A-1-as TAATATATCGAAGGTATGTGT
(SEQ. ID. No. 111) CFTR14A-2-s GAGCATACCAGCAGTGACTACA (SEQ. ID. No.
112) CFTR14A-2-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGTAATACTTTA
CAATAGAACATTCTTACC (SEQ. ID. No. 113) CFTR14A-3-s
ACCAGCAGTGACTACATGGA (SEQ. ID. No. 114) CFTR14A-3-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATATTTATGTGTG
TGCATATATATGTAT (SEQ. ID. No. 115) CFTR14B-1-s
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGTGTACCTTG ATATTGG (SEQ.
ID. No. 116) CFTR14B-1-as CTCACTTTCCAAGGAG (SEQ. ID. No. 117)
CF14B-3-s GGTGTGGCTCCTTGG (SEQ. ID. No. 118) CF14B-3-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGACTACAGC CCTGAACTCC (SEQ.
ID. No. 119) CFTR15A-s
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCATGTATTGGAA ATTCAGTAAGTAAC
(SEQ. ID. No. 120) CFTR15A-as TTCGACACTGTGATTAGAGTATGC (SEQ. ID.
No. 121) alternate 15B: CF15B-s2 CGTGGGAGTAGCCGAC (SEQ. ID. No.
122) CF15B-as2 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCATTAGAAA
ACCAACAAA (SEQ. ID. No. 123) CF16A-s5
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCTGAATG CGTCTACTG (SEQ. ID.
No. 124) CF16A-as5 CATCCAAAATTGCTATA (SEQ. ID. No. 125) CFTR16B-s
TTGAGGAATTTGTCATCTTGTAT (SEQ. ID. No. 126) CFTR16B-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCAAAAT- CACA TTTGCTTTTGTTA
(SEQ. ID. No. 127) CF17A-1-s6
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGAAAGAAATA AATCACTGA (SEQ.
ID. No. 128) CF17A-1-as6 GTAAAACTGCGACAAC (SEQ. ID. No. 129)
CFTR17A-2-s CCAACATGTTTTCTTTGATCTTACAG (SEQ. ID. No. 130)
CFTR17A-2-as CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCC- GAGAATCTC
AAATAGCTCTTATAGCTTT (SEQ. ID. No. 131) CFTR17B-1-s
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTTAACCAATGA CATTTGTGATA
(SEQ. ID. No. 132) CFTR17B-1-as GTGTCCATAGTCCTTTTAAGC (SEQ. ID. No.
133) CFTR17B-2-s
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATATTTCACAGG CAGGAGTCC
(SEQ. ID. No. 134) CFTR17B-2-as AAAATCATTTCTATTCTCATTTGGA (SEQ. ID.
No. 135) CFTR17B-3-s ACTTCGTGCCTTCGGAC (SEQ. ID. No. 136)
CFTR17B-3-as CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGC- CCCCGCCCGCAGCAATGAAG
AAGATGACAAA (SEQ. ID. No. 137) CFTR17B-4-s CTGGTTCCAAATGAGAA (SEQ.
ID. No. 138) CFTR17B-4-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTAACCTATAGAA TGCAGCA (SEQ.
ID. No. 139) CFTR18A-s
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTTAATGTGATA TGTGCCCTA (SEQ.
ID. No. 140) CFTR18A-as AGATGATAAGACTTACCAAGC (SEQ. ID. No. 141)
CFTR18B-s GAGAAGGAGAAGGAAGAGTTG (SEQ. ID. No. 142) CFTR18B-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGC- CCGCTTCCTCATGCT ATTACTCATAC
(SEQ. ID. No. 143) CFTR19A-s2
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGAAGTTATTTTTTA GGAAGCAT
(SEQ. ID. No. 144) CFTR19A-as GAACTTAAAGACTCGGCTC (SEQ. ID. No.
145) CFTR19B-s
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCGGCCCGGAAATTGTCTGCC ATTCTTAA
(SEQ. ID. No. 146) CFTR19B-as GAGTTGGCCATTCTTGTATG (SEQ. ID. No.
147) CF19C-s3 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCC-
GCCCGTGTGAGCCGAGT CTTT (SEQ. ID. No. 148) CF19C-as2
ATGGCATTTCCACCTT (SEQ. ID. No. 149) CF19D-s2 CGTGAAGAAAGATGAC (SEQ.
ID. No. 150) CF19D-as2
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTAATGTTACAAA TAGATTC (SEQ.
ID. No. 151) CF19i-s2 CGCCCGCCGCGCCCCGCGCCCGCCCCGCC-
GCCCCCGCCCGCTTGATTTCTG GAGAC (SEQ. ID. No. 152) CF19i-as2
CTAGCTGTAATTGCAT (SEQ. ID. No. 153) new 20-s:
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCTGAATTATGTTT ATGGCA (SEQ.
ID. No. 154) new 20-as CGTTTTTTCTGGCTAAGT (SEQ. ID. No. 155)
alternate 21A CF21A-s3
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGAGTTATTCATACT TTCTTCT (SEQ.
ID. No. 156) CF21A-as3 AGCCTTACCTCATCTG (SEQ. ID. No. 157) CF21B-s3
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCC- GTTTCTGGAACA TTTAG (SEQ.
ID. No. 158) CF21B-as3 GAATGATGTCAGCTATAT (SEQ. ID. No. 159)
CFTR22A-s2 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGAGCTGTCA
AGGTTGTA (SEQ. ID. No. 160) CFTR22A-as2 CAGGAAACTGTTCTATCAC (SEQ.
ID. No. 161) CFTR22B-s CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCC-
CGGAATGTCAACTGC TTGAGTGTTTT (SEQ. ID. No. 162) CFTR22B-as
AAGTAACAGAACATCTGAAACTCACAC (SEQ. ID. No. 163) CFTR22C-s2
CTTGCTGCTTGATGAAC (SEQ. ID. No. 164) CFTR22C-as
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGGGCAATTATTT CATATCTTGG
(SEQ. ID. No. 165) CF23A-s3
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTATCAAGGTAA ATACAGA (SEQ.
ID. No. 166) CF23A-as3 GCTTCTATCCTGTGTTC (SEQ. ID. No. 167)
CF23B-s2 GATATTATGTGTGGTATTTTC (SEQ. ID. No. 168) CF24A-s2
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGAACTTGTACA TTGTTGCA (SEQ.
ID. No. 169) CF24A-s2
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCCTTTGAGCC TGTGCC (SEQ. ID.
No. 170) CF24A-as2 GCTTGAGTTCCGGTGG (SEQ. ID. No. 171) CF24B-s2
CATCAGCCCCTCCGAC (SEQ. ID. No. 172) CF2413-s2
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTTTCTGAGG CAGAGGTA (SEQ.
ID. No. 173)
[0134]
3TABLE 3 TTGE Group Listing for CFTR Gene Gene Fragment Group
Position run group PCR temp CFTR 16A-5 1 A 45.5-52.5 49 CFTR 6B2 1
B 125 V 49 CFTR 17B1 1 C 1.5 rr 52 CFTR 21A3 1 D run time 4.67
hours 52 CFTR 12 2 A 60 CFTR 5A 2 B 52 CFTR 7A 2 C 62 CFTR 16B 3 A
49 CFTR 7D 3 B 60 CFTR 5B 3 C 62 CFTR 17B4 4 A 49 CFTR 6B1 4 B 54
CFTR 6A3-2 4 C 52 CFTR 8B2 5 A 47.5-53 52 CFTR 2B5 5 B 150 V 49
CFTR 13A 6 A 1 rr 59 CFTR 8A 6 B run time 5.5 hours 60 CFTR 11A 7 A
50.5-56.5 49 CFTR 19A 7 B 125 V 54 CFTR 19B 7 C 1.5 rr 59 CFTR
14B2-3 8 A run time 4 hours 60 CFTR 13B3 8 B 49 CFTR 21B3 8 C 46
CFTR 14A3 9 A 54 CFTR 17A2 9 B 60 CFTR 4B 9 C 52 CFTR 13F2 10 A 46
CFTR 23A3 10 B 46 CFTR 19in 10 C 49 CFTR 14A2 10 D 62 CFTR 3A2 11 A
50.5-56.5 46 CFTR 18A 11 B 125 V 60 CFTR 2A 11 C 1.2 rr 54 CFTR 10
12 A run time 5 hours 59 CFTR 14A1 12 B 46 CFTR 22A2 12 C 52 CFTR
10C3 13 A 50.5-56.5 54 CFTR 11B 13 B 125 V 52 CFTR 3B 14 A 1.5 rr
54 CFTR 18B 14 B run time 4 hours 60 CFTR 17A16 14 C 52 CFTR 9Ts 15
A 50.5-56.5 59 CFTR 9C 15 B 150 V 59 prerun 30 min CFTR 23B2 16 A
1.2 rr 49 CFTR 13C 16 B run time 5 hours 60 CFTR 22C 17 A 54.5-61
59 CFTR 22B 17 B 125 V 59 CFTR 6A2 17 C 1.5 rr 49 CFTR 15A 18 A run
time 4 hours 54 CFTR 19D2 18 B 46 CFTR 4A 18 C 60 CFTR 14B1 18 D 54
CFTR 15B2 19 A 54 CFTR 17B3 19 B 60 CFTR 6A1 19 C 62 CFTR 17B2 20 A
49 CFTR 19C3 20 B 52 CFTR 24B2 20 C 52 CFTR 20 21 A 55-60 49 CFTR
7C 21 B 150 V, 60 1 rr, run time 5 hours CFTR 13E 22 A 59-64 59
CFTR 1A 22 B 125 V 59 CFTR 24A2 23 A 1.5 rr 62 CFTR 1B 23 B run
time 3.3 hours 59
[0135] Although the invention has been described with reference to
embodiments and examples, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims.
4TABLE A CF exon 1 541 gggaggggtg ctggcggggg tgcgtagtgg gtggagaaag
ccgctagagc aaatttgggg (SEQ. ID. NO. 174) 601 ccggaccagg cagcactcgg
cttttaacct gggcagtgaa ggcgggggaa agagcaaaag 661 gaaggggtgg
tgtgcggagt aggggtgggt ggggggaatt ggaagccaaa tgacatcaca 721
gcaggtcaga gaaaaagggt tgagcggcag gcacccagag tagtaggtct ttggcattag
781 gagcttgagc ccagacggcc ctagcaggga ccccagcgcc cagagaccAT
GCAGAGGTCG 841 CCTCTGGAAA AGGCCAGCGT TGTCTCCAAA CTTTTTTTCA
Ggtgagaagg tggccaaccg 901 agcttcggaa agacacgtgc ccacgaaaga
ggagggcgtg tgtatgggtt gggtttgggg 961 taaaggaata agcagttttt
aaaaagatgc gctatcattc attgttttga aagaaaatgt 1021 gggtattgta
gaataaaaca gaaagcatta agaagagatg gaagaatgaa ctgaagctga 1081
ttgaatagag agccacatct acttgcaact gaaaagttag aatctcaaga ctcaagtacg
1141 ctactatgca cttgttttat ttcatttttc taagaaacta aaaatacttg
ttaataagta CFTR1A-s: 5' CGCCCCCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG-
(SEQ. ID. NO. 175) GGAAGCCAAATGACATCACAGC 3' CFTR1A-as: 5'
TGAAAAAAACTTTGGAGACAACGC 3' (SEQ. ID. NO. 176) CFTR1B-s: 5'
CCCAGCGCCCAGAGACC 3' (SEQ. ID. NO. 177) CFTR1B-as: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 178)
ACTGCTTATTCCTTTACCCCAA 3' CFTR1-s-tag: 5' GGGTGGTGTGCGGAGTA 3'
(SEQ. ID. NO. 179) CFTR1-as-tag: 5' CAAAACAATGAATGATAGCG 3' (SEQ.
ID. NO. 180) CF exon 2 1 aaaccatact attattccct cccaatccct
ttgacaaagt gacagtcaca ttagttcaga (SEQ. ID. NO. 181) 61 gatattgatg
ttttatacag gtgtagcctg taagagatga agcctggtat ttatagaaat 121
tgacttattt tattctcata tttacatgtg cataattttc catatgccag aaaagttgaa
181 tagtatcaga ttccaaatct gtatggagac caaatcaagt gaatatctgt
tcctcctctc 241 tttattttag CTGGACCAGA CCAATTTTGA GGAAAGCATA
CAGACAGCGC CTGGAATTGT 301 CAGACATATA CCAAATCCCT TCTGTTCATT
CTGCTCACAA TCTATCTGAA AAATTGGAAA 361 Ggtatgttca tgtacattgt
ttagttgaag agagaaattc atattattaa ttatttagag 421 aagagaaagc
aaacatatta taagtttaat tcttatattt aaaaatagga gccaagtatg 481
gtggctaatg cctgtaatcc caactatttg ggaggccaag atgagaggat tgcttgagac
541 caggagtttg ataccagcct gggcaacata gcaagatgtt atctctacac
aaaataaaaa 601 gttagctggg aatggtagtg catgcttgta CF2A-s: 5'
CGCCCGCCGCGCCCCGCGCCCGCC- CCGCCGCCCCCGCCCG- (SEQ. ID. NO. 182)
CCAGAAAAGTTGAATAGTATCAG 3' CF2A-as: 5' AGATTGTCAGCAGAATCAA 3' (SEQ.
ID. NO. 183) CFTR2-s-tag: 5' GACAGTCACATTAGTTCAG 3' (SEQ. ID. NO.
184) CFTR2-as-tag: 5' TGTTTGCTTTCTCTTCT 3' (SEQ. ID. NO. 185)
CF2B-s5: 5' ATACCAAATCCCTTCTG 3' (SEQ. ID. NO. 186) CF2B-as5: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 187)
TGCTTTCTCTTCTCTAAAT 3' CF exon 3 1 aggaatctgc cagatatctg gctgagtgtt
tggtgttgta tggtctccat gagattttgt (SEQ. ID. NO. 188) 61 ctctataata
cttgggttaa tctccttgga tatacttgtg tgaatcaaac tatgttaagg 121
gaaataggac aactaaaata tttgcacatg caacttattg gtcccacttt ttattctttt
181 gcagAGAATG GGATAGAGAG CTGGCTTCAA AGAAAAATCC TAAACTCATT
AATGCCCTTC 241 GGCGATGTTT TTTCTGGAGA TTTATGTTCT ATGGAATCTT
TTTATATTTA GGGgtaagga 301 tctcatttgt acattcatta tgtatcacat
aactatatgc atttttgtga ttatgaaaag 361 actacgaaat ctggtgaata
ggtgtaaaaa tataaaggat gaatccaact ccaaacacta 421 agaaaccacc
taaaactcta gtaaggataa gtaa CF3A-s: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 189)
TGGTGTTGTATGGTCT 3' CF3A-as: 5' AACATAAATCTCCAGAA 3' (SEQ. ID. NO.
190) CF3B-s: 5' GCTGGCTTCAAAGAAAAATCC 3' (SEQ. ID. NO. 191)
CF3B-as: 5' CGCCCGCCGCGCCCCGCGCCCGCCCC- CCCGCCCCCGCCCG- (SEQ. ID.
NO. 192) CACCAGATTTCGTAGTCTTTTCA 3' CFTR-3-s-tag: 5'
TGGTGTTGTATGGTCTC 3' (SEQ. ID. NO. 193) CFTR-3-as-tag: 5'
TTAGGTCCTTTCTTAGTG 3' (SEQ. ID. NO. 194) CF exon 4 1 ccactattca
ctgtttaact taaaatacct catatgtaaa cttgtctccc actgttgcta (SEQ. ID.
NO. 195) 61 taacaaatcc caagtcttat ttcaaagtac caagatattg aaaatagtgc
taagagtttc 121 acatatggta tgaccctcta tataaactca ttttaagtct
cctctaaaga tgaaaagtct 181 tgtgttgaaa ttctcagggt attttatgag
aaataaatga aatttaattt ctctgttttt 241 ccccttttgt agGAAGTCAC
CAAAGCAGTA CAGCCTCTCT TACTCCCAAG AATCATAGCT 301 TCCTATGACC
CGGATAACAA GGAGGAACGC TCTATCCCGA TTTATCTAGG CATAGGCTTA 361
TGCCTTCTCT TTATTGTGAG GACACTGCTC CTACACCCAG CCATTTTTGG CCTTCATCAC
421 ATTGGAATGC AGATGAGAAT AGCTATCTTT AGTTTGATTT ATAAGAAGgt
aatacttcct 481 tgcacaggcc ccatggcaca tatattctgt atcgtacatg
ttttaatgtc ataaattagg 541 tagtgagctg gtacaagtaa gggataaatg
ctgaaattaa tttaatatgc ctattaaata 601 aatggcagga ataattaatg
ctcttaatta tccttgataa tttaattgac ttaaactgat 661 aattattgag tatc
CF4A-s: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO.
196) AATTTCTCTGTTTTTCCCCTT 3' CF4A-as: 5' AGCTATTCTCATCTGCATTCCA 3'
(SEQ. ID. NO. 197) CF4B-s: 5' GACACTCCTCCTACACC 3' (SEQ. ID. NO.
198) CF4B-as: 5' CGCCCGCCGCGCCCCCCCCCCGCCCCGCCGCCCCCGCCCG- (SEQ.
ID. NO. 199) TCAGCATTTATCCCTTA 3' CF4-s-tag: 5' ATAACAAATCCCAAGTC
3' (SEQ. ID. NO. 200) CF4-as-tag: 5' TGTACCAGCTCACTACC 3' (SEQ. ID.
NO. 201) CF exon 5 1 taattatttc tgcctagatg ctgggaaata aaacaactag
aagcatgcca gtataatatt (SEQ. ID. NO. 202) 61 gactgttgaa agaaacattt
atgaacctga gaagatagta agctagatga atagaatata 121 attttcatta
cctttactta ataatgaatg cataataact gaattagtca tattataatt 181
ttacttataa tatatttgta ttttgtttgt tgaaattatc taactttcca tttttctttt
241 agACTTTAAA GCTGTCAAGC CCTGTTCTAG ATAAAATAAC TATTGGACAA
CTTGTTAGTC 301 TCCTTTCCAA CAACCTCAAC AAATTTGATG AAgtatgtac
ctattgattt aatcttttag 361 gcactattgt tataaattat acaactggaa
aggcggagtt ttcctgggtc agataatagt 421 aattagtggt taagtcttgc
tcagctctag cttccctatt ctggaaacta agaaaggtca 481 attgtatagc
agagcaccat tctggggtct ggtagaacca cccaactcaa aggcacctta 541
gcctgttgtt aataagattt ttcaaaactt aattcttatc agaccttgct tcttttaaac
CF5A-s: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO.
203) ATAATATATTTGTATTTTGTTTGTTG 3' CF5A-as: 5' AATTTGTTCAGGTTGTTGGA
3' (SEQ. ID. NO. 204) CF5B-s: 5' AGCTGTCAAGCCGTGTTC 3' (SEQ. ID.
NO. 205) CF5B-as: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG-
(SEQ. ID. NO. 206) ATCTGACCCAGGAAAACTC 3' CF5-s-tag: 5'
TGCTGGGAAATAAAAC 3' (SEQ. ID. NO. 207) CF5-as-tag: 5'
AGAATGGTGCTCTGCT 3' (SEQ. ID. NO. 208) CF exon 6A 1 gacatgatac
ttaagatgtc caatcttgat tccactgaat aaaaatatgc ttaaaaatgc (SEQ. ID.
NO. 209) 61 actgacttga aatttgtttt ttgggaaaac cgattctatg tgtagaatgt
ttaagcacat 121 tgctatgtgc tccatgtaat gattacctag attttagtgt
gctcagaacc acgaagtgtt 181 tgatcatata agctcctttt acttgctttc
tttcatatat gattgttagt ttctaggggt 241 ggaagataca atgacacctg
tttttgctgt gcttttattt tccagGGACT TGCATTGGCA 301 CATTTCGTGT
GGATCGCTCC TTTGCAACTC GCACTCCTCA TGGGGCTAAT CTGGGAGTTG 361
TTACAGGCGT CTGCCTTCTG TGGACTTGGT TTCCTGATAG TCCTTGCCCT TTTTCAGGCT
421 GGGCTAGGGA GAATGATGAT GAAGTACAGg tagcaaccta ttttcataac
ttgaaagttt 481 taaaaattat gttttcaaaa agcccacttt agtaaaacca
ggactgctct atgcatagaa 541 cagtgatctt cagtgtcatt aaattttttt
tttttttttt tttgagacag agtctagatc 601 tgtcacccag gctggagtgc
agtggcacga tcttggctca ctgcactgca acttctgcct 661 cccaggctca
agcaattctc ctgcctcagc ctccggagta gctgggatta gaggcgcatg CF6A-1-s: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 210)
TTGTTAGTTTCTAGGGGTGG 3' CF6A-1-as: 5' AAGGACTATCAGGAAACCAAG 3'
(SEQ. ID. NO. 211) CF6A-2-s: 5' GCTAATCTGGGAGTTGTTAC 3' (SEQ. ID.
NO. 212) CF6A-2-as: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCG-
(SEQ. ID. NO. 213) AGTTATGAAAATAGGTTGCTAC 3' CF6A-3-s2:
5'CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGGGAGAATGAT- (SEQ. ID.
NO. 214) GATGAAG 3' CF6A-3-as2: 5'ACACTGAAGATCACTGTTCTA 3' (SEQ.
ID. NO. 215) CF6a-s-tag: 5' CTCCTTTTACTTGCTTTC 3' (SEQ. ID. NO.
216) CF6a-as-tag: 5' GAGCACTCCTGGTTTTA 3' (SEQ. ID. NO. 217) CF
exon 6B
atgagtctgtacagcgtctggcacataggaggcatttaccaaacagtagttattatttttgttaccatcta
(SEQ. ID. NO. 218) tttgataataaaataatgcccatctgttgaataaaag-
aaatatgacttaaaaccttgagcagttcttaata gataatttgacttgtttttacta-
ttagattgattgattgattgattgattgatttacagAGATCAGAGAGC
TGGGAAGATCAGTGAAAGACTTGTGATTACCTCAGAAATGATTGAAAATATCCAATCTGTTAAGGCATACT
GCTGGGAAGAAGCAATGGAAAAAATGATTGAAAACTTAAGACAgtaagttgttccaata-
atttcaatattg ttagtaattctgtccttaattttttaaaaatatgtttatcatggt-
agacttccacctcatatttgatgttt gtgacaatcaaatgattgcatttaagttctg-
tcaatattcatgcattagttgca CF6B-1-s: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 219)
CCTTGAGCAGTTCTTAATAGATA 3' CF6B-1-as: 5' ATGCCTTAACAGATTGGATAT 3'
(SEQ. ID. NO. 220) CF6B-2-s: 5' GAAAATATCCAATCTGTTAAG 3' (SEQ. ID.
NO. 221) CF6B-2-as: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG-
(SEQ. ID. NO. 222) TGAGGTGGAAGTCTACCA 3' CF6b-s-tag: 5'
AAAACCTTGAGCAGTT 3' (SEQ. ID. NO. 223) CF6b-as-tag: 5'
GGTGGAAGTCTACCATG 3' (SEQ. ID. NO. 224) CF exon 7 1 tttacaagta
ctacaagcaa aacactggta ctttcattgt tatcttttca tataaggtaa (SEQ. ID.
NO. 225) 61 ctgaggccca gagagattaa ataacatgcc caaggtcaca caggtcatat
gatgtggagc 121 caggttaaaa atataggcag aaagactcta gagaccatgc
tcagatcttc cattccaaga 181 tccctgatat ttgaaaaata aaataacatc
ctgaatttta ttgttattgt tttttatagA 241 ACAGAACTGA AACTGACTCG
GAAGGCAGCC TATGTGAGAT ACTTCAATAG CTCAGCCTTC 301 TTCTTCTCAG
GGTTCTTTGT GGTGTTTTTA TCTGTGCTTC CCTATGCACT AATCAAAGGA 361
ATCATCCTCC GGAAAATATT CACCACCATC TCATTCTCCA TTGTTCTGCG CATGGCGGTC
421 ACTCGGCAAT TTCCCTGGGC TGTACAAACA TGGTATGACT CTCTTGGAGC
AATAAACAAA 481 ATACAGgtaa tgtaccataa tgctgcatta tatactatga
tttaaataat cagtcaatag 541 atcagttcta atgaactttg caaaaatgtg
cgaaaagata gaaaaagaaa tttccttcac 601 taggaagtta taaaagttgc
cagctaatac taggaatgtt caccttaaac ttttcctagc 661 atttctctgg
acagtatgat ggatgagagt ggcatttatg caaattacct taaaatccca 721
ataatactga tgtagctagc agctttgaga aa CF7A-s: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 226)
AGACCATGCTCAGATCTTCCATT 3' CF7A-as: 5' GCTGCCTTCCGAGTCAGTTTCAGT 3'
(SEQ. ID. NO. 227) CF7C-s: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 228)
ACTGAAACTGACTCGGAAGG 3' CF7C-as: 5' ATGGTACATTACCTGTATTTTGTTTA 3'
(SEQ. ID. NO. 229) CF7D-s: 5' CTGTACAAACATGGTATGACTCTCTT 3' (SEQ.
ID. NO. 230) CF7D-as: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG-
(SEQ. ID. NO. 231) GTGAAGGAAATTTCTTTTTCTATCT 3' CF7-s-tag: 5'
AATATAGGCAGAAAGACT 3' (SEQ. ID. NO. 232) CF7-as-tag: 5'
GAACTGATCTATTGACTGA 3' (SEQ. ID. NO. 233) CF exon 8 1 gcacattagt
gggtaattca gggttgcttt gtaaattcat cactaaggtt agcatgtaat (SEQ. ID.
NO. 234) 61 agtacaagga agaatcagtt gtatgttaaa tctaatgtat aaaaagtttt
ataaaatatc 121 atatgtttag agagtatatt tcaaatatga tgaatcctag
tgcttggcaa attaacttta 181 gaacactaat aaaattattt tattaagaaa
taattactat ttcattatta aaattcatat 241 ataagatgta gcacaatgag
agtataaagt agatgtaata atgcattaat gctattctga 301 ttctataata
tgtttttgct ctcttttata aatagGATTT CTTACAAAAG CAAGAATATA 361
AGACATTGGA ATATAACTTA ACGACTACAG AAGTAGTGAT GGAGAATGTA ACAGCCTTCT
421 GGGAGGAGgt cagaattttt aaaaaattgt ttgctctaaa cacctaactg
ttttcttctt 481 tgtgaatatg gatttcatcc taatggcgaa taaaattaga
atgatgatat aactggtaga 541 actggaagga ggatcactca cttattttct
agattaagaa gtagaggaat ggccaggtgc 601 tcatggttgt aatcccagca
ctttcgggag accaaggcgg gtggatcacc tgaggtcagg 661 agttcaagac
cagcctgcca acatggtaaa acccggtctc tactaaaaat acaaaaaatt 721 aactg
CF8A-s: 5' CGCCCGCCGCGCCCCGCGCCCGCC- CCGCCGCCCCCGCCCG- (SEQ. ID.
NO. 235) GCACAATGAGAGTATAAAGTAG 3' CF8A-as: 5'
CCATCACTACTTCTGTAGTCG 3' (SEQ. ID. NO. 236) CF8B-s2: 5'
CTCTCTTTTATAAATAGGATTTCTTAC 3' (SEQ. ID. NO. 237) CF8B-as2: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 238)
TTCCAGTTCTACCAGTTATATCATC 3' CF8-s-tag: 5' ATGAATCCTAGTGCTTG 3'
(SEQ. ID. NO. 239) CF8-as-tag: 5' TCCTTCCAGTTCTACC 3' (SEQ. ID. NO.
240) CF exon 9 181 tgtatgtgta tgtatacatg tatgtattca gtctttactg
aaattaaaaa atctttaact (SEQ. ID. NO. 241) 241 tgataatggg caaatatctt
agttttagat catgtcctct agaaaccgta tgctatataa 301 ttatgtacta
taaagtaata atgtatacag tgtaatggat catgggccat gtgcttttca 361
aactaattgt acataaaaca agcatctatt gaaaatatct gacaaactca tcttttattt
421 ttgatgtgtg tgtgtgtgtg tgtgtgtgtt tttttaacag GGATTTGGGG
AATTATTTGA 481 GAAAGCAAAA CAAAACAATA ACAATAGAAA AACTTCTAAT
GGTGATGACA GCCTCTTCTT 541 CAGTAATTTC TCACTTCTTG GTACTCCTGT
CCTGAAAGAT ATTAATTTCA AGATAGAAAG 601 AGGACAGTTG TTGGCGGTTG
CTGGATCCAC TGGAGCAGGC AAGgtagttc ttttgttctt 661 cactattaag
aacttaattt ggtgtccatg tctctttttt tttctagttt gtagtgctgg 721
aaggtatttt tggagaaatt cttacatgag cattaggaga atgtatgggt gtagtgtctt
781 gtataataga aattgttcca ctgataattt actctagttt tttatttcct
catattattt 841 tcagtggctt tttcttccac atctttatat tttgcaccac
attcaacact gtatcttgca 901 catggcgagc attcaataac tttattgaat
aaacaaatca tccattttat ccattcttaa 961 ccagaacaga cattttttca
gagctggtcc aggaaaatca tgacttacat tttgccttag 1021 taaccacata
aacaaaaagt ctccattttt gttgac CF9C-s: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 242)
ACAATAGAAAAACTTCTAATGGTGA 3' CF9C-as: 5' AAAAAAGAGACATGGACACCAA 3'
(SEQ. ID. NO. 243) CF9-s-tag: 5' AGAAACCGTATGCTAT 3' (SEQ. ID. NO.
244) CF9-as-tag: 5' CCCATACATTCTCCTA 3' (SEQ. ID. NO. 245)
CF9-as2-tag: 5' TAAAGATGTGGAAGAAA 3' (SEQ. ID. NO. 246) CF exon 10
1 cactgtagct gtactacctt ccatctcctc aacctattcc aactatctga atcatgtgcc
(SEQ. ID. NO. 247) 61 cttctctgtg aacctctatc ataatacttg tcacactgta
ttgtaattgt ctcttttact 121 ttcccttgta tcttttgtgc atagcagagt
acctgaaaca ggaagtattt taaatatttt 181 gaatcaaatg agttaataga
atctttacaa ataagaatat acacttctgc ttaggatgat 241 aattggaggc
aagtgaatcc tgagcgtgat ttgataatga cctaataatg atgggtttta 301
tttccagACT TCACTTCTAA TGATGATTAT GGGAGAACTG GAGCCTTCAG AGGGTAAAAT
361 TAAGCACAGT GGAAGAATTT CATTCTGTTC TCAGTTTTCC TGGATTATGC
CTGGCACCAT 421 TAAAGAAAAT ATCATCTTTG GTGTTTCCTA TGATGAATAT
AGATACAGAA GCGTCATCAA 481 AGCATGCCAA CTAGAAGAGg taagaaacta
tgtgaaaact ttttgattat gcatatgaac 541 ccttcacact acccaaatta
tatatttggc tccatattca atcggttagt ctacatatat 601 ttatgtttcc
tctatgggta agctactgtg aatggatcaa ttaataaaac acatgaccta 661
tgctttaaga agcttgcaaa cacatgaaat aaatgcaatt tattttttaa ataatgggtt
721 catttgatca caataaatgc attttatgaa atggtgagaa ttttgttcac
tcattagtga 781 gacaaacgtc tcaatggtta tttatatggc atgcatatag
tgatatgtgg t CF10-s: 5' CCTGAGCGTGATTTGATA 3' (SEQ. ID. NO. 248)
CF10-as: 5' CGCCCGCCGCGCCCCGCGCCCGCCC- CGCCGCCCCCGCCCG- (SEQ. ID.
NO. 249) ATGTAGACTAACCGATTGAA 3' CF10C-s: 5' GGGAGAACTGGAGCCT 3'
(SEQ. ID. NO. 250) CF10C-as: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 251)
AACCGATTGAATATGGAG 3' CF10-s2-tag: 5' CCTTGTATCTTTTGTGC 3' (SEQ.
ID. NO. 252) CF10-as2-tag: 5' CCGATTGAATATGGAG 3' (SEQ. ID. NO.
253) CF exon 11 1 atatacccat aaatatacac atattttaat ttttggtatt
ttataattat tatttaatga (SEQ. ID. NO. 254) 61 tcattcatga cattttaaaa
attacaggaa aaatttacat ctaaaatttc agcaatgttg 121 tttttgacca
actaaataaa ttgcatttga aataatggag atgcaatgtt caaaatttca 181
actgtggtta aagcaatagt gtgatatatg attacattag aaggaagatg tgcctttcaa
241 attcagattg agcatactaa aagtgactct ctaattttct atttttggta
atagGACATC 301 TCCAAGTTTG CAGAGAAAGA CAATATAGTT CTTGGAGAAG
GTGGAATCAC ACTGAGTGGA 361 GGTCAACGAG CAAGAATTTC TTTAGCAAGg
tgaataacta attattggtc tagcaagcat 421 ttgctgtaaa tgtcattcat
gtaaaaaaat tacagacatt tctctattgc tttatattct 481 gtttctggaa
ttgaaaaaat cctggggttt tatggctagt gggttaagaa tcacatttaa 541
gaactataaa taatggtata gtatccagat ttggtagaga ttatggttac tcagaatctg
601 tgcccgtatc ttgg CF11A-s: 5'
CGCCCCCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 255)
GATATATGATTACATTAGAAG 3' CF11A-as: 5' ACCTTCTCCAAGAACTA 3' (SEQ.
ID. NO. 256) CF11B-s: 5' ATAGGACATCTCCAAGTT 3' (SEQ. ID. NO. 257)
CF11B-as: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID.
NO. 258) GCAATAGAGAAATGTCTGT 3' CF11-s-tag: 5' CAGATTGAGCATACTAAAAG
3' (SEQ. ID. NO. 259) CF11-as-tag: 5' AAGATACGGGCACAGA 3' (SEQ. ID.
NO. 260) CF exon 12 1 cttacagtta gcaaaatcac ttcagcagtt cttggaatgt
tgtgaaaagt gataaaaatc (SEQ. ID. NO. 261) 61 ttctgcaact tattccttta
ttcctcattt aaaataatct accatagtaa aaacatgtat 121 aaaagtgcta
cttctgcacc acttttgaga atagtgttat ttcagtgaat cgatgtggtg 181
accatattgt aatgcatgta gtgaactgtt taaggcaaat catotacact agatgaccag
241 gaaatagaga ggaaatgtaa tttaatttcc attttctttt tagAGCAGTA
TACAAAGATG 301 CTGATTTGTA TTTATTAGAC TCTCCTTTTG GATACCTAGA
TGTTTTAACA GAAAAAGAAA 361 TATTTGAAAG gtatgttctt tgaatacctt
acttataatg ctcatgctaa aataaaagaa 421 agacagactg tcccatcata
gattgcattt tacctcttga gaaatatgtt caccattgtt 481 ggtatggcag
aatgtagcat ggtattaact caaatctgat ctgccctact gggccaggat 541
tcaagattac ttccattaaa accttttctc accgcctcat gctaaaccag tttctctcat
601 tgctatactg ttatagcaat tgctatctat gtagtttttg cagtatcatt
gccttgtgat 661 atatattact ttaatt CF12-s: 5'
CGCCCGCCGCGCCCCGCGCCCGCC- CCGCCGCCCCCGCCCG- (SEQ. ID. NO. 262)
GTGAACTGTTTAAGGCAAATCAT 3' CF12-as: 5' TGATGGGACAGTCTGTCTTTC 3'
(SEQ. ID. NO. 263) CF12-s-tag: 5' TCACTTCAGCAGTTCTT 3' (SEQ. ID.
NO. 264) CF12-as-tag: 5' CAATCTATGATGGGACA 3' (SEQ. ID. NO. 265) CF
exon 13 1 gaattcacaa ggtaccaatt taattactac agagtactta tagaatcatt
taaaatataa (SEQ. ID. NO. 266) 61 taaaattgta tgatagagat tatatgcaat
aaaacattaa caaaatgcta aaatacgaga 121 catattgcaa taaagtattt
ataaaattga tatttatatg tttttatatc ttaaagCTGT 181 GTCTGTAAAC
TGATGGCTAA CAAAACTAGG ATTTTGGTCA CTTCTAAAAT GGAACATTTA 241
AAGAAAGCTG ACAAAATATT AATTTTGCAT GAAGGTAGCA GCTATTTTTA TGGGACATTT
301 TCAGAACTCC AAAATCTACA GCCAGACTTT AGCTCAAAAC TCATGGGATG
TGATTCTTTC 361 GACCAATTTA GTGCAGAAAG AAGAAATTCA ATCCTAACTG
AGACCTTACA CCGTTTCTCA 421 TTAGAAGGAG ATGCTCCTCT CTCCTGGACA
GAAACAAAAA AACAATCTTT TAAACAGACT 481 GGAGAGTTTG GGGAAAAAAG
GAAGAATTCT ATTCTCAATC CAATCAACTC TATACGAAAA 541 TTTTCCATTG
TGCAAAAGAC TCCCTTACAA ATGAATGGCA TCGAAGAGGA TTCTGATGAG 601
CCTTTAGAGA GAAGGCTGTC CTTAGTACCA GATTCTGAGC AGGGAGAGGC GATACTGCCT
661 CGCATCAGCG TGATCAGCAC TGGCCCCACG CTTCAGGCAC GAAGGAGGCA
GTCTGTCCTG 721 AACCTGATGA CACACTCAGT TAACCAAGGT CAGAACATTC
ACCGAAAGAC AACAGCATCC 781 ACACGAAAAG TGTCACTGGC CCCTCAGGCA
AACTTGACTG AACTGGATAT ATATTCAAGA 841 AGGTTATCTC AAGAAACTGG
CTTCGAAATA AGTGAAGAAA TTAACCAAGA AGACTTAAAG 901 gtaggtatac
atcgcttggg ggtatttcac cccacagaat gcaattgagt agaatgcaat 961
atgtagcatg taacaaaatt tactaaaatc ataggattag gataaggtgt atcttaaaac
1021 tcagaaagta tgaagttcat taattataca agcaacgtta aaatgtaaaa
taacaaatga 1081 tttctttttg caatggacat atctcttccc ataaaatggg
aaaggattta gtttttggtc 1141 ctctactaag ccagtgataa ctgtgactat
agttagaaag catttgcttt attaccatct CFTR13A-s: 5'
AATACGAGACATATTGCAATAAAGT 3' (SEQ. ID. NO. 267) CFTR13A-as: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 268)
CTGGCTGTAGATTTTGGAGTTC 3' CF13B-s3: 5' AGGTAGCAGCTATTTTT 3' (SEQ.
ID. NO. 269) CF13B-as3: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 270)
GGACAGCCTTCTCTCTA 3' CFTR13C-s: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATGGGACATTTTC- (SEQ. ID.
NO. 271) AGAACTCC 3' CFTR13C-as: 5' CCTCTTCGATGCCATTCAT 3' (SEQ.
ID. NO. 272) CFTR13D-s: 5' CGCCCGCCGCGCCCCGCGCCCGCC-
CCGCCGCCCCCGCCCGCAATCCAATCAAC- (SEQ. ID. NO. 273) TCTATACGAA 3'
CFTR13D-as: 5' CTGATCACGCTGATGCGA 3' (SEQ. ID. NO. 274) CFTR13E-s:
5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGATGAGC- C (SEQ. ID.
NO. 275) TTTAGAGAGAA 3' CFTR13E-as: 5' CCAGTTCAGTCAAGTTTGC 3' (SEQ.
ID. NO. 276) CF13F-s2: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCAGCGTGAT- (SEQ. ID. NO.
277) CAGCA 3' CF13F-as2: 5' TTTGTTACATGCTACATA 3' (SEQ. ID. NO.
278) CF exon 14A 1 ggaaacttca tttagatggt atcattcatt tgataaaagg
tatgccactg ttaagccttt (SEQ. ID. NO. 279) 61 aatggtaaaa ttgtccaata
ataatacagt tatataatca gtgatacatt tttagaattt 121 tgaaaaatta
cgatgtttct catttttaat aaagctgtgt tgctccagta gacattattc 181
tggctataga atgacatcat acatggcatt tataatgatt tatatttgtt aaaatacact
241 tagattcaag taatactatt cttttatttt catatattaa aaataaaacc
acaatggtgg 301 catgaaactg tactgtctta ttgtaatagc cataattctt
TTATTCAGGA GTGCTTTTTT 361 GATGATATGG AGAGCATACC AGCAGTGACT
ACATGGAACA CATACCTTCG ATATATTACT 421 GTCCACAAGA GCTTAATTTT
TGTGCTAATT TGGTGCTTAG TAATTTTTCT GGCAGAGgta 481 agaatgttct
attgtaaagt attactggat ttaaagttaa attaagatag tttggggatg 541
tatacatata tatgcacaca cataaatatg tatatataca catgtataca tgtataagta
601 tgcatatata cacacatata tcactatatg tatatatgta tatattacat
atatttgtga 661 ttttacagta tataatggta tagattcata tagttcttag
cttctgaaaa atcaacaagt 721 agaaccacta ctga CFTR14A-1-s: 5'
CGCCCGCCGCCCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 280)
TTCATATATTAAAAATAAAACC 3' CFTR14A-1-as: 5' TAATATATCGAAGGTATGTGT 3'
(SEQ. ID. NO. 281) CFTR14A-2-s: 5' GAGCATACCAGCAGTGACTACA 3' (SEQ.
ID. NO. 282) CFTR14A-2-as: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 283)
GTAATACTTTACAATAGAACATTCTTACC 3' CFTR14A-3-s: 5'
ACCAGCAGTGACTACATGGA 3' (SEQ. ID. NO. 284) CFTR14A-3-as: 5'
ATATTTATGTGTGTGCATATATATGTAT 3' (SEQ. ID. NO. 285) CF14A-s-tag: 5'
TGTTGCTCCAGTAGACA 3' (SEQ. ID. NO. 286) CF14A-as-tag: 5'
CATCCCCAAACTATCT 3' (SEQ. ID. NO. 287) CF exon 14B 1 gaattccatt
aacttaatgt ggtctcatca caaataatag tacttagaac acctagtaca (SEQ. ID.
NO. 288) 61 gctgctggac ccaggaacac aaagcaaagg aagatgaaat tgtgtgtacc
ttgatattgg 121 tacacacatc aaatggtgtg atgtgaattt agatgtgggc
atgggaggaa taggtgaaga 181 tgttagaaaa aaaatcaact gtgtcttgtt
ccattccagG TGGCTGCTTC TTTGGTTGTG 241 CTGTGGCTCC TTGGAAAgtg
agtattccat gtcctattgt gtagattgtg ttttatttct 301 gttgattaaa
tattgtaatc cactatgttt gtatgtattg taatccactt tgtttcattt 361
ctcccaagca ttatggtagt ggaaagataa ggttttttgt ttaaatgatg accattagtt
421 gggtgaggtg acacattcct gtagtcctag ctcctccaca ggctgacgca
ggaggatcac 481 ttgagcccag gagttcaggg ctgtagtgtt gtatcattgt
gagtagccac caccgcactc 541 cagcctggac aatatagtga gatcctatat
ctaaaataaa ataaaataaa atgaataaat 601 tgtgagcatg tgcagctcct g
CFTR14B-1-s: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID.
NO. 289) GTGTACCTTGATATTGG 3' CFTR14B-1-as: 5' CTCACTTTCCAAGGAG 3'
(SEQ. ID. NO. 290) CF14B-3-s: 5' GCTGTGGCTCCTTGG 3' (SEQ. ID. NO.
291) CF14B-3-as: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCC- GCCCCCGCCCG-
(SEQ. ID. NO. 292) ACTACAGCCCTGAACTCC 3' CF14B-s-tag: 5'
GGAACACAAAGCAAAG 3' (SEQ. ID. NO. 293) CF14B-as-tag: 5'
TGGGAGAAATGAAACA 3' (SEQ. ID. NO. 294) CF exon 15 1 tcctatatct
aaataaataa ataaatgaat aaattgtgag catgtgcagc tcctgcagtt (SEQ. ID.
NO. 295) 61 tctaaagaat atagttctgt tcagtttctg tgaaacacaa taaaaatatt
tgaaataaca 121 ttacatattt agggttttct tcaaattttt taatttaata
aagaacaact caatctctat 181 caatagtgag aaaacatatc tattttcttg
caataatagt atgattttga ggttaagggt 241 gcatgctctt ctaatgcaaa
atattgtatt tatttagact caagtttagt tccatttaca 301 tgtattggaa
attcagtaag taactttggc tgccaaataa cgatttccta tttgctttac 361
agCACTCCTC TTCAAGACAA AGGGAATAGT ACTCATAGTA GAAATAACAG CTATGCACTG
421 ATTATCACCA GCACCAGTTC GTATTATGTG TTTTACATTT ACGTGGGAGT
AGCCGACACT 481 TTGCTTGCTA TGGGATTCTT CAGACGTCTA CCACTGGTGC
ATACTCTAAT CACAGTGTCG 541 AAAATTTTAC ACCACAAAAT GTTACATTCT
GTTCTTCAAG CACCTATGTC AACCCTCAAC 601 ACGTTGAAAG CAGgtacttt
actaggtcta agaaatgaaa ctgctgatcc accatcaata 661 gggcctgtgg
ttttgttggt tttctaatgg cagtgctggc ttttgcacag aggcatgtgc 721 ctttgtt
CFTR15A-s: 5'CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCATGTATTGGAA
(SEQ. ID. NO. 296) ATTCAGTAAGTAAC 3' CFTR15A-as:
5'TTCGACACTGTGATTAGAGTATGC 3' (SEQ. ID. NO. 297) CFTR15B-s: 5'
GTGGGAGTAGCCGACA 3' (SEQ. ID. NO. 298) CFTR15B-as: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 299)
CAGGCCCTATTGATGGT 3' CF15B-s2: 5' CGTGGGAGTAGCCGAC 3' (SEQ. ID. NO.
300) CF15B-as2: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ.
ID. NO. 301) CATTAGAAAACCAACAAA 3' CF15-s-tag: 5'
AGACTCAAGTTTAGTTCCA 3' (SEQ. ID. NO. 302) CF15-as-tag: 5'
CCAACAAAACCACAGG 3' (SEQ. ID. NO. 303) CF exon 16 1 gtaagattgt
aagcaggatg agtacccacc tattcctgac ataatttata gtaaaagcta (SEQ. ID.
NO. 304) 61 tttcagagaa attggtcgtt acttgaatct tacaagaatc tgaaactttt
aaaaaggttt 121 aaaagtaaaa gacaataact tgaacacata attatttaga
atgtttggaa agaaacaaaa 181 atttctaagt ctatctgatt ctatttgcta
attcttattt gggttctgaa tgcgtctact 241 gtgatccaaa cttagtattg
aatatattga tatatcttta aaaaattagt gttttttgag 301 gaatttgtca
tcttgtatat tatagGTGGG ATTCTTAATA GATTCTCCAA AGATATAGCA 361
ATTTTGGATG ACCTTCTGCC TCTTACCATA TTTGACTTCA TCCAGgtatg taaaaataag
421 taccgttaag tatgtctgta ttattaaaaa aacaataaca aaagcaaatg
tgattttgtt 481 ttcatttttt atttgattga gggttgaagt cctgtctatt
gcattaattt tgtaattatc 541 caaagccttc aaaatagaca taagtttagt
aaattcaata ataagtcaga actgcttacc 601 tggcccaaac ctgaggcaat
cccacattta gatgtaatag ctgtctactt gggagtgatt 661 tgagaggcac
aaaggaccat ctttcccaaa atcactggca c CF16A-s5
5'CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCTGAATGCGTCT- (SEQ. ID.
NO. 305) ACTG 3' CF16A-as5 5'CATCCAAAATTGCTATA 3' (SEQ. ID. NO.
306) CFTR16B-s: 5' TTGAGGAATTTGTCATCTTGTAT 3' (SEQ. ID. NO. 307)
CFTR16B-as: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID.
NO. 308) CAAAATCACATTTGCTTTTGTTA 3' CF16-s-tag: 5'
ATGCGTCTACTGTGATC 3' (SEQ. ID. NO. 309) CF16-as-tag: 5'
CTTCAACCCTCAATCA 3' (SEQ. ID. NO. 310) CF exon 17A 1 agtgcaccag
catggcacat gtatacatat gtaactaacc tcgacaatgt gcacatgtac (SEQ. ID.
NO. 311) 61 cctaaaactt aaagtataat aaaaaaaata aaaaaaagtt tgaggtgttt
aaagtatgca 121 aaaaaaaaaa aagaaataaa tcactgacac actttgtcca
ctttgcaatg tgaaaatgtt 181 tactcaccaa catgttttct ttgatcttac
agTTGTTATT AATTGTGATT GGAGCTATAG 241 CAGTTGTCGC AGTTTTACAA
CCCTACATCT TTGTTGCAAC AGTGCCAGTG ATAGTGGCTT 301 TTATTATGTT
GAGAGCATAT TTCCTCCAAA CCTCACAGCA ACTCAAACAA CTGGAATCTG 361
AAGgtatgac agtgaatgtg cgatactcat cttgtaaaaa agctataaga gctatttgag
421 attctttatt gttaatctac ttaaaaaaaa ttctgctttt aaacttttac
atcatataac 481 aataattttt ttctacatgc atgtgtatat aaaaggaaac
tatattacaa agtacacatg 541 gatttttttt cttaattaat gaccatgtga
cttcattttg gttttaaaat aggtatatag 601 aatcttacca cagttggtgt
acaggacatt catttat CF17A-1-s6: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 312)
AAAGAAATAAATCACTGA 3' CF17A-1-as6: 5' GTAAAACTGCGACAAC 3' (SEQ. ID.
NO. 313) CFTR17A-2-s: 5' CCAACATGTTTTCTTTGATCTTACAG 3' (SEQ. ID.
NO. 314) CFTR17A-2-as: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG-
(SEQ. ID. NO. 315) AGAATCTCAAATAGCTCTTATAGCTTT 3' CF17A-s-tag: 5'
AAATAAATCACTGACACAC 3' (SEQ. ID. NO. 316) CF17A-as-tag: 5'
AATGAAGTCACATGGTC 3' (SEQ. ID. NO. 317) CF exon 17B 1 ttcaaagaat
ggcaccagtg tgaaaaaaag ctttttaacc aatgacattt gtgatatgat (SEQ. ID.
NO. 318) 61 tattctaatt tagtcttttt caggtacaag atattatgaa aattacattt
tgtgtttatg 121 ttatttgcaa tgttttctat ggaaatattt cacagGCAGG
AGTCCAATTT TCACTCATCT 181 TGTTACAAGC TTAAAAGGAC TATGGACACT
TCGTGCCTTC GGACGGCAGC CTTACTTTGA 241 AACTCTGTTC CACAAAGCTC
TGAATTTACA TACTGCCAAC TGGTTCTTGT ACCTGTCAAC 301 ACTGCGCTGG
TTCCAAATGA GAATAGAAAT GATTTTTGTC ATCTTCTTCA TTGCTGTTAC 361
CTTCATTTCC ATTTTAACAA CAGgtactat gaactcatta actttagcta agcatttaag
421 taaaaaattt tcaatgaata aaatgctgca ttctataggt tatcaatttt
tgatatcttt 481 agagtttagt aattaacaaa tttgttggtt tattattgaa
caagtgattt ctttgaaatt 541 tccattgttt tattgttaaa caaataattt
ccttgaaatc ggtatatata tatatatagt CFTR17B-1-s: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 319)
TTAACCAATGACATTTGTGATA 3' CFTR17B-1-as: 5' GTGTCCATAGTCCTTTTAAGC 3'
(SEQ. ID. NO. 320) CF17B-2-s2: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 321)
AATATTTCACAGGCAG 3' CF17B-2-as2: 5' TGAAGGTAACAGCAAT 3' (SEQ. ID.
NO. 322) CFTR17B-3-s: 5' ACTTCGTGCCTTCGGAC 3' (SEQ. ID. NO. 323)
CFTR17B-3-as: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ.
ID. NO. 324) CAGCAATGAAGAAGATGACAAA 3' CFTR17B-4-s: 5'
CTGGTTCCAAATGAGAA 3' (SEQ. ID. NO. 325) CFTR17B-4-as: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 326)
TAACCTATAGAATGCAGCA 3' CF exon 18 1 ttattactta tagaataata
gtagaagaga caaatatggt acctacccat taccaacaac (SEQ. ID. NO. 327) 61
acctccaata ccagtaacat tttttaaaaa gggcaacact ttcctaatat tcaatcgctc
121 tttgatttaa aatcctggtt gaatacttac tatatgcaga gcattattct
attagtagat 181 gctgtgatga actgagattt aaaaattgtt aaaattagca
taaaattgaa atgtaaattt 241 aatgtgatat gtgccctagg agaagtgtga
ataaagtcgt tcacagaaga gagaaataac 301 atgaggttca tttacgtctt
ttgtgcatct atagGAGAAG GAGAAGGAAG AGTTGGTATT 361 ATCCTGACTT
TAGCCATGAA TATCATGAGT ACATTGCAGT GGGCTGTAAA CTCCAGCATA 421
GATGTGGATA GCTTGgtaag tcttatcatc tttttaactt ttatgaaaaa aattcagaca
481 agtaacaaag tatgagtaat agcatgagga agaactatat accgtatatt
gagcttaaga 541 aataaaacat tacagataaa ttgagggtca ctgtgtatct
gtcattaaat ccttatctct 601 tctttccttc toatagatag ccactatgaa
gatctaatac tgcagtgagc attctttcac 661 ctgtttcctt attcaggatt
ttctaggaga aatacctagg ggttgtattg ctgggtcata 721 ggattcaccc
atgcttaac CFTR18A-s: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG-
(SEQ. ID. NO. 328) TTAATGTGATATGTGCCCTA 3' CFTR18A-as: 5'
AGATGATAAGACTTACCAAGC 3' (SEQ. ID. NO. 329) CFTR18B-s: 5'
GAGAAGGAGAAGGAAGAGTTG 3' (SEQ. ID. NO. 330) CFTR18B-as: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 331)
CTTCCTCATGCTATTACTCATAC 3' CF18-s-tag: 5' CCTGGTTGAATACTTACT 3'
(SEQ. ID. NO. 332) CF18-as-tag: 5' CTCATACTTTGTTACTTGTC 3' (SEQ.
ID. NO. 333) CF exon 19 1 ttctcttcag ttaaactttt aattatatcc
aattatttcc tgttagttca ttgaaaagcc (SEQ. ID. NO. 334) 61 cgacaaataa
ccaagtgaca aatagcaagt gttgcatttt acaagttatt ttttaggaag 121
catcaaacta
attgtgaaat tgtctgccat tcttaaaaac aaaaatgttg ttatttttat 181
ttcagATGCG ATCTGTGAGC CGAGTCTTTA AGTTCATTGA CATGCCAACA GAAGGTAAAC
241 CTACCAAGTC AACCAAACCA TACAAGAATG GCCAACTCTC GAAAGTTATG
ATTATTGAGA 301 ATTCACACGT GAAGAAAGAT GACATCTGGC CCTCAGGGGG
CCAAATGACT GTCAAAGATC 361 TCACAGCAAA ATACACAGAA GCTGGAAATG
CCATATTAGA GAACATTTCC TTCTCAATAA 421 GTCCTGGCCA GAGGgtgaga
tttgaacact gcttgctttg ttagactgtg ttcagtaagt 481 gaatcccagt
agcctgaagc aatgtgttag cagaatctat ttgtaacatt attattgtac 541
agtagaatca atattaaaca cacatgtttt attatatgga gtcattattt ttaatatgaa
601 atttaatttg cagagtctga actatatat CF19A-s2: 5'
CGCCCGCCGCGCCCCGCGCCCGC- CCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 335)
AAGTTATTTTTTAGGAAGCAT 3' CF19A-as: 5' GAACTTAAAGACTCGGCTC 3' (SEQ.
ID. NO. 336) CF19B-s: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG-
(SEQ. ID. NO. 337) GAAATTGTCTGCCATTCTTAA 3' CF19B-as: 5'
GAGTTGGCCATTCTTGTATG 3' (SEQ. ID. NO. 338) CF19C-s3: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 339)
TGTGAGCCGAGTCTTT 3' CF19C-as2: 5' ATGGCATTTCCACCTT 3' (SEQ. ID. NO.
340) CF19D-s2: 5' CGTGAAGAAAGATGAC 3' (SEQ. ID. NO. 341) CF19D-as2:
5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCG- CCCCCGCCCG- (SEQ. ID. NO. 342)
TAATGTTACAAATAGATTC 3' CF19-s-tag: 5' GACAAATAACCAAGTGAC 3' (SEQ.
ID. NO. 343) CF19-as-tag: 5' AACACATTGCTTCAGG 3' (SEQ. ID. NO. 344)
CF intron 19 29941 acttaactgc tttctccatt tgtagtctct tgaaaataca
gaaatttcag aaataattta (SEQ. ID. NO. 345) 30001 taagaatatc
aaggattcaa atcatatcag cacaaacacc taaatacttg tttgctttgt 30061
taaacacata tcccattttc tatcttgata aacattggtg taaagtagtt gaatcattca
30121 gtgggtataa gcagcatatt ctcaatacta tgtttcatta ataattaata
gagatatatg 30181 aacacataaa agattcaatt ataatcacct tgtggatcta
aatttcagtt gacttgtcat 30241 cttgatttct ggagaccaca aggtaatgaa
aaataattac aagagtcttc catctgttgc 30301 agtattaaaa tggcgagtaa
gacaccctga aaggaaatgt tctattcatg gtacaatgca 30361 attacagcta
gcaccaaatt caacactgtt taactttcaa catattattt tgatttatct 30421
tgatccaaca ttctcaggga ggaggtgcat tgaagttatt agaaaacact gacttagatt
30481 tagggtatgt cttaaaagct tatttgcggg aagtactcta gccttattca
acagateact 30541 gagaagcctg gaaaaacaaa tcccggaaac taattattat
gtgccagtta tataaacaag 30601 aagactttgt tgggtacaaa ccagtgattc
cttgcctttg aaaaatgtgt cagatatcat CF19i-s2:
5'CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCTTGATTTCTG- (SEQ. ID.
NO. 346) GAGAC 3' CF19i-as2: 5'CTAGCTGTAATTGCAT 3' (SEQ. ID. NO.
347) CF19i-s1 tag: 5'tagAGTGGGTATAAGCAGC 3' (SEQ. ID. NO. 348)
CF19i-as1 tag: 5'tagGTTGAATAAGGCTAGAGTA 3' (SEQ. ID. NO. 349) CF
exon 20 1 aaaggtcagt gataaaggaa gtctgcatca ggggtccaat tccttatggc
cagtttctct (SEQ. ID. NO. 350) 61 attctgttcc aaggttgttt gtctccatat
atcaacattg gtcaggattg aaagtgtgca 121 acaaggtttg aatgaataag
tgaaaatctt ccactggtga caggataaaa tattccaatg 181 gtttttattg
aagtacaata ctgaattatg tttatggcat ggtacctata tgtcacagaa 241
gtgatcccat cacttttacc ttatagGTGG GCCTCTTGGG AAGAACTGGA TCAGGGAAGA
301 GTACTTTGTT ATCAGCTTTT TTGAGACTAC TGAACACTGA AGGAGAAATC
CAGATCGATG 361 GTGTGTCTTG GGATTCAATA ACTTTGCAAC AGTGGAGGAA
AGCCTTTGGA GTGATACCAC 421 AGgtgagcaa aaggacttag ccagaaaaaa
ggcaactaaa ttatattttt tactgctatt 481 tgatacttgt actcaagaaa
ttcatattac tctgcaaaat atatttgtta tgcattgctg 541 tctttttttt
ctccagtgca gttttctcat aggcagaaaa gatgtctcta aaagtttggg CFTR20-s: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 351)
GAATTATGTTTATGGCATGGT 3' CFTR20-as: 5' GAGTACAAGTATCAAATAGCAGTAA 3'
(SEQ. ID. NO. 352) CF20-s-tag: 5' AAATCTTCCACTGGTGA 3' (SEQ. ID.
NO. 353) CF20-as-tag: 5' GACATCTTTTCTGCCTAT 3' (SEQ. ID. NO. 354)
new 20-s: 5'CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCTGAATTAT-
(SEQ. ID. NO. 355) GTTTATGGCA3' new 20-as: 5'CCTTTTTTCTGGCTAAGT3'
(SEQ. ID. NO. 356) CF exon 21 1 tttttaatat tctacaatta acaattatct
caatttcttt attctaaaga cattggatta (SEQ. ID. NO. 357) 61 gaaaaatgtt
cacaagggac tccaaatatt gctgtagtat ttgtttctta aaagaatgat 121
acaaagcaga catgataaaa tattaaaatt tgagagaact tgatggtaag tacatgggtg
181 tttcttattt taaaataatt tttctacttg aaatatttta caatacaata
agggaaaaat 241 aaaaagttat ttaagttatt catactttct tcttcttttc
ttttttgcta tagAAAGTAT 301 TTATTTTTTC TGGAACATTT AGAAAAAACT
TGGATCCCTA TGAACAGTGG AGTGATCAAG 361 AAATATGGAA AGTTGCAGAT
GAGgtaaggc tgctaactga aatgattttg aaaggggtaa 421 ctcataccaa
cacaaatggc tgatatagct gacatcattc tacacacttt gtgtgcatgt 481
atgtgtgtgc acaactttaa aatggagtac cctaacatac ctggagcaac aggtactttt
541 gactggacct acccctaact gaaatgattt tgaaagaggt aactcatacc
aacacaaatg 601 gttgatatgg ctaagatcat tctacacact ttgtgtgcat
gtatttctgt gcacaacttc 661 aaaatggagt accctaaaat acctggcgcg
acaagtactt ttgactgagc ctactt CF21A-s2: 5' ATGGTAAGTACATGGGTGTT 3'
(SEQ. ID. NO. 358) CF21A-as2: 5' CCACTGTTCATAGGGATCCAAG 3' (SEQ.
ID. NO. 359) CF21B-s3: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG-
(SEQ. ID. NO. 360) TTTCTGGAACATTTAG 3' CF21B-as3: 5'
GAATGATGTCAGCTATAT 3' (SEQ. ID. NO. 361) CF21-s-tag: 5'
TGTTCACAAGGGACTC 3' (SEQ. ID. NO. 362) CF21-as-tag: 5'
CAGTTAGGGGTAGGTC 3' (SEQ. ID. NO. 363) CF 21A-s3: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 364)
AGTTATTCATACTTTCTTCT 3' CF21A-as3: 5' AGCCTTACCTCATCTG 3' (SEQ. ID.
NO. 365) CF exon 22 1 cacagttgac tattttatgc tatcttttgt cctcagtcat
gacagagtag aagatgggag (SEQ. ID. NO. 366) 61 gtagcaccaa ggatgatgtc
atacctccat cctttatgct acattctatc ttctgtctac 121 ataagatgtc
atactagagg gcatatctgc aatgtataca tattatcttt tccagcatgc 181
attcagttgt gttggaataa tttatgtaca cctttataaa cgctgagcct cacaagagcc
241 atgtgccacg tattgtttct tactactttt ggatacctgg cacgtaatag
acactcattg 301 aaagtttcct aatgaatgaa gtacaaagat aaaacaagtt
atagactgat tcttttgagc 361 tgtcaaggtt gtaaatagac ttttgctcaa
tcaattcaaa tggtggcagg tagtgggggt 421 agagggattg gtatgaaaaa
cataagcttt cagaactcct gtgtttattt ttagaatgtc 481 aactgcttga
gtgtttttaa ctctgtggta tctgaactat cttctctaac tgcagGTTGG 541
GCTCAGATCT GTGATAGAAC AGTTTCCTGG GAAGCTTGAC TTTGTCCTTG TGGATGGGGG
601 CTGTGTCCTA AGCCATGGCC ACAAGCAGTT GATGTGCTTG GCTAGATCTG
TTCTCAGTAA 661 GGCGAAGATC TTGCTGCTTG ATGAACCCAG TGCTCATTTG
GATCCAGTgt gagtttcaga 721 tgttctgtta cttaatagca cagtgggaac
agaatcatta tgcctgcttc atggtgacac 781 atatttctat taggctgtca
tgtctgcgtg tgggggtctc ccaagatatg aaataattgc 841 ccagtggaaa
tgagcataaa tgcatatttc cttgctaaga gttcttgtgt tttcttccga 901
agatagtttt CFTR22A-s2: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCG-
CCGCCCCCGCCCG- (SEQ. ID. NO. 367) TGAGCTGTCAAGGTTGTA 3'
CFTR22A-as2: 5' CAGGAAACTGTTCTATCAC 3' (SEQ. ID. NO. 368)
CFTR22B-s: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID.
NO. 369) GAATGTCAACTGCTTGAGTGTTTT 3' CFTR22B-as: 5'
AAGTAACAGAACATCTGAAACTCACAC 3' (SEQ. ID. NO. 370) CF22C-s: 5'
CTTGCTGCTTGATGAAC 3' (SEQ. ID. NO. 371) CF22C-as: 5'
GCAATTATTTCATATCTTGG 3' (SEQ. ID. NO. 372) CF22-s-tag: 5'
AGGGATTGGTATGAAAA 3' (SEQ. ID. NO. 373) CF22-as-tag: 5'
GGAAGAAAACACAAGAAC 3' (SEQ. ID. NO. 374) CF exon 23 1 gcatgtttat
agccccaaat aaaagaagta ctggtgattc tacataatga aaatgtactc (SEQ. ID.
NO. 375) 61 atttattaaa gtttctttga aatatttgtc ctgtttattt atggatactt
agagtctacc 121 ccatggttga aaagctgatt gtgcgtaacg ctatatcaac
attatgtgaa aagaacttaa 181 agaaataagt aatttaaaga gataatagaa
caatagacat attatcaagg taaatacaga 241 tcattactgt tctgtgatat
tatgtgtggt attttctttc ttttctagAA CATACCAAAT 301 AATTAGAAGA
ACTCTAAAAC AAGCATTTGC TGATTGCACA GTAATTCTCT GTGAACACAG 361
GATAGAAGCA ATGCTGGAAT GCCAACAATT TTTGgtgagt ctttataact ttacttaaga
421 tctcattgcc cttgtaattc ttgataacaa tctcacatgt gatagttcct
gcaaattgca 481 acaatgtaca agttcttttc aaaaatatgt atcatacagc
catccagctt tactcaaaat 541 agctgcacaa gtttttcact ttgatctgag
ccatgtggtg aggttgaaat atagtaaatc 601 taaaatggca gcatattact
aagttatgtt tataaatagg atatatatac ttttgagccc 661 tttatttggg
accaagtcat acaaaatact ctactgttta agattttaaa aaaggtccct 721
gtgattcttt caataactaa atgtcccatg gatgtggtct ggacaggcct agttgtctta
781 cagtctgatt tatggtatta atgacaaagt tgagaggcac atttcatttt
tctagccatg CF23A-s3: 5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG-
(SEQ. ID. NO. 376) TATCAAGGTAAATACAGA 3' CF23A-as3: 5'
GCTTCTATCCTGTGTTC 3' (SEQ. ID. NO. 377) CF23B-s2: 5'
GATATTATGTGTGGTATTTTC 3' (SEQ. ID. NO. 378) CF23B-as2:
5'CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGAACTTGTACA (SEQ. ID. NO.
379) TTGTTGCA 3' CF exon 24 1 agatggtaga acctccttag agcaaaagga
cacagcagtt aaatgtgaca tacctgattg (SEQ. ID. NO. 380) 61 ttcaaaatgc
aaggctctgg acattgcatt ctttgacttt tattttcctt tgagcctgtg 121
ccagtttctg tccctgctct ggtctgacct gccttctgtc ccagatctca ctaacagcca
181 tttccctagG TCATAGAAGA GAACAAAGTG CGGCAGTACG ATTCCATCCA
GAAACTGCTG 241 AACGAGAGGA GCCTCTTCCG GCAAGCCATC AGCCCCTCCG
ACAGGGTGAA GCTCTTTCCC 301 CACCGGAACT CAAGCAAGTG CAAGTCTAAG
CCCCAGATTG CTGCTCTGAA AGAGGAGACA 361 GAAGAAGAGG TGCAAGATAC
AAGGCTTTAG agagcagcat aaatgttgac atgggacatt 421 tgctcatgga
attggagctc gtgggacagt cacctcatgg aattggagct cgtggaacag 481
ttacctctgc ctcagaaaac aaggatgaat taagtttttt tttaaaaaag aaacatttgg
541 taaggggaat tgaggacact gatatgggtc ttgataaatg gcttcctggc
aatagtcaaa 601 ttgtgtgaaa ggtacttcaa atccttgaag atttaccact
tgtgttttgc aagccagatt 661 ttcctgaaaa cccttgccat gtgctagtaa
ttggaaaggc agctctaaat gtcaatcagc CF24A-s2: 5'
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 381)
CCTTTGAGCCTGTGCC 3' CF24A-as2: 5' GCTTGAGTTCCGGTGG 3' (SEQ. ID. NO.
382) CF24B-s2: 5' CATCAGCCCCTCCGAC 3' (SEQ. ID. NO. 383) CF24B-s2:
5' CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG- (SEQ. ID. NO. 384)
TTTCTGAGGCAGAGGTA 3' CF24-s-tag: 5' GCAGTTAAATGTGACATACC 3' (SEQ.
ID. NO. 385) CF24-as-tag: 5' TCCTTGTTTTCTGAGGC 3' (SEQ. ID. NO.
386)
[0136]
5TABLE B Alignment to GemBank Accession Number AH006034 primer
alignment locus upstre downstr. on AH006034 primer name start end
50 bp 50 bp Primer sequences 5'-3' HUMCFTRA1 CFTR1A-s 701 722 651
772 CGCCCGCCGCGCCCCGCGCCCGCCCCG- CCGCCCCCGCCCGGGAAGCAAATGACATCACAGC
(Seq Id No. 387) CFTR1B-as 857 880 807 903 TGAAAAAAAAGTTTTGGAGCACAF
(Seq Id No. 388) CFTR2A-s2 167 189 117 239 CCAGCGCCAGAGACC (Seq Id
No. 389) CFTR1B-as 955 976 905 1026
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGACTGCT- TATTCCTTTACCCCAA
(Seq Id No. 390) HUMCFTRA2 CRTR2A-sa 167 189 117 239
GGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCCAGAAAAGTTGAATAGTATC- AG
(Seq Id No. 391) CFTR2A-as2 325 343 275 393 AGATTGTCAGCAGAATCAA
(Seq Id No. 392) CF2B-s5: 308 324 258 374 ATACCAAATCCCTTCTG (Seq Id
No. 393) CF2B-as5: 413 431 363 481
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCGTGCTTTCTCTTCTCTAAT (Seq Id
No. 394) CFTR2B-s2 92 312 242 362 CGCCCGCCGCGCCCCGCGCCCGCCCC-
GCCGCCCCCGCCCGTGGAATTGTCAGACATATACC (Seq Id No. 395) CFTR2B-as2 470
486 420 536 AGCCACCATACTTGGCT (Seq Id No. 396) HUMCFTRA3 CF3A-s2 31
46 -19 96 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTG-
GTGTTGTATGGTCT (Seq Id No. 397) CF3A-as2 252 268 202 318
AACATAAATCTCCAGAA (Seq Id No. 398) CFTR3A-s 58 77 8 127
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGTCTCTATAATACTTGGGT (Seq
Id No. 399) CFTR3A-as 266 287 216 337 ATATAAAAAGATTCCATAGAAC (Seq
Id No. 400) CFTR3B-s 200 220 150 270 GCTGGCTTCAAAGAAAAATCC (Seq Id
No. 401) CFTR3B-as 354 376 304 426 CGCCCGCCGCGCCCCGCGCCCGCCCCG-
CCGCCCCCGCCCGCACCAGATTTCGTAGTCTTTTCA (Seq Id No. 402) HUMCFTRA4
CFTR4A-s 226 246 176 296 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCC-
CGAATTTCTCTGTTTTTCCCCTT (Seq Id No. 403) CFTR4A-as 423 444 373 494
AGCTATTCTCATCTGCATTCCA (Seq Id No. 404) CFTR4B-s 381 397 331 447
GACACTGCTCCTACACC (Seq Id No. 405) CFTR4B-as 557 574 507 624
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGTCAGCATTTATCCCTTA (Seq Id No.
406) HUMCETRA5 CFTR5A-s 187 212 137 262
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATAATATATTTGTATTTTGTTTGTTG
(Seq Id No. 407) CFTRSA-as 306 325 256 375 AATTTGTTCAGGTTGTTGGA
(Seq Id No. 408) CFTR5B-a 250 267 200 317 AGCTGTCAASCCGTGTTC (Seq
Id No. 409) CFTR5B-as 396 414 346 464
CGCCCGCCGCCCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATCTGACCCAGCAAAACTC (Seq Id
No. 410) HUMCFTRA6 CFTR6A-1-a 223 242 173 292
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTTGTTAGTTTCTAGGGTGG (Seq Id
No. 411) CFTR6A-1-as 385 405 335 455 AAGGACTATCACGAAACCAAG (Seq Id
No. 412) CFTR6A-2-s 345 364 295 414 GCTAATCTCCGAGTTGTTAC (Seq Id
No. 413) CFTR6A-2-as 450 471 400 521
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGAGTTATGAAAATAGGTTGCTAC (Seq
Id No. 414) CF6A-3-s 427 444 377 494
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGGAGAATGATGATGAAG (Seq Id
No. 415) CF6A-3-as 536 556 486 606 ACACTGAAGATCACTGTTCTA (Seq Id
No. 416) CFTR6A-3-s 401 418 351 468 TCCTTGCCCTTTTTCAGG (Seq Id No.
417) CFTR6A-3-as 539 553 489 613 CGCCCGCCGCGCCCCGCGCCCGCCC-
CGCCGCCCCCGCCCCTTTAATGACACTGAAGATCACTGTT (SEQ Id No. 418)
CFTR6B-1-s2 1504 1526 1454 1576
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCCC- CTTGAGCAGTTCTTAATAGATA
(Seq Id No. 419) CFTR6B-1-as2 1641 1661 1591 1711
ATGCCTTAACAGATTGGATAT (Seq Id No. 420) CFTR6B-2-s2 1637 1657 1567
1707 GAAAATATCCAATCTGTTAAG (Seq Id No. 421) CFTR6B-2-as2 1777 1794
1727 1844 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCC-
GCCCGTGAGGTGGAGTCTACCA (Seq Id No. 422) CFTR6B-2-s 1637 1652 1587
1702 GAAAATATCCAATCTG (Seq Id No. 423) CFTR6B-2-as 1780 1795 1730
1845 CGCCCGCCGCGCCCCGCGCCCGCCCCGCC~CCCCCGCCCGATGAGGTGGAAGTCTA (Seq
Id No. 424) HUMCFTRA7 CPTR7A-s 152 174 102 224
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGAGACCATGCTCAGATCTTCCATT
(Seq Id No. 425) CFTR7A-as 246 269 196 319 GCTGCCTTCCGAGTCAGTTTCAGT
(Seq Id No. 426) CFTR7C-s 246 265 196 315
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGACTCCGGAAGG (Seq Id No.
427) CFTR7C-as 476 498 426 548 ATGGTACATTACCTGTATTTTGTTTA (Seq Id
No. 428) CFTR7D-s 440 465 390 515 CTGTACAAACATGGTATGACTCTCTT (Seq
Id No. 429) CFTR7D-as 576 600 526 650 CGCCCGCCGCGCCCCGCGCCCGCCCCG-
CCGCCCCCGCCCGGTGAAGGAAATTTCTTTTTCTATCT (Seq Id No. 430) CFTR7B-s
152 175 102 225 AGACCATGCTCAGATCTTCCATTC (Seq Id No. 431) CFTR7B-as
246 267 196 317 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGCCTT-
CCGAGTCAGTTTCAGT (Seq Id No. 432) HUMCFTRA8 CFTR8A-s 251 272 201
322 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGCACAATGAGAGTATAAAGTA-
G (Seq Id No. 433) CFTR8A-as 382 402 332 452 CCATCACTACTTCTGTAGTCG
(Seq Id No. 434) CF8B-S2: 319 345 269 395
CTCTCTTTTATAAATAGGATTTCTTAC (Seq Id No. 435) CF8B-as2 523 547 473
597
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTTCCAGTTCTACCAGTTATATCATC
(Seq Id No.436) CFTR8B-s 319 345 269 395
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCTCTCTTTTATAAATAGGATTTCTTAC
(Seq Id No. 437) CSTR8B-as 523 547 473 597
TTCCAGTTCTACCAGTTATATCATC (Seq Id No. 438) HUMCFTRA9 CFTR9C-s 501
525 451 575
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGACAATAGAAAAACTTCTAATGGTGA
(Seq Id No. 439) CFTR9C-as 679 700 629 750 AAAAAAGAGACATGGACACCAA
(Seq Id No. 440) HUMCFTRA10 CFTR10-s 259 276 209 326
CCTGAGCGTGATTTGATA (Seq Id No. 441) CFTR10-ae 331 346 281 396
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATGTAGACTAACCGATTGAA (Seq
Id No. 442) CF10C-e3 333 346 283 396 GGGAGAACTGGAGCCT (Seq Id No.
443) CF10C-as3 357 587 520 637 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCG-
CCCCCGCCCGCCCGATTGAATATGGAG (Seq Id No. 444) HUMCFTRA11 CFTR11A-s2
203 223 153 273 CGCCCGCCGCGCCCGCGCCCGCCCCGCCGCCCCCGCCGGATATATG-
ATTACATTAGAAG (Seq Id No. 445) CFTR11A-as2 326 342 276 392
ACCTTCTCCAAGAACTA (Seq Id No. 446) CFTR11B-s 291 308 241 358
ATAGGACATCTCCAAGTT (Seq Id No. 447) CFTR11B-as 452 470 402 520
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGCAATAGAGAAATGTCTGT (Seq Id
No. 448) HUMCFTRA12 CFTR12-s 201 223 151 273
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGTGTTTAAGGCAATCAT (Seq Id
No. 449) CFTR12-as 418 438 368 488 TGATGGGACAGTCTGTCTTTC (Seq Id
No. 450) HUMCFTRA13 CFTR13A-s 112 136 62 186
AATACGAGACATATTGCAATAAAGT (Seq Id No. 451) CFTR13A-as 304 325 254
375 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCTGGCTGTAGATTTTGGAGTTC
(Seq Id No. 452) CF13B-s 273 289 223 339 AGGTAGCAGCTATTTTT (Seq Id
No. 455) CF13B-as 360 621 555 671 CGCCCGCCGCGCCCCGCGCCCGC-
CCCGCCGCCCCCGCCCGGGACAGCCTTCTCTCTA (Seq Id No. 456) CFTR13B-s 291
243 169 293 CACTTCTAAAATGGAACATTTAAAG (Seq Id No. 457) CFTR13B-as
641 658 591 708 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGCA-
GTATCGCCTCTCCCT (Seq Id No. 458) CFTR13C-s 290 310 240 360
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATGGGACATTTTCAGAACTCC (Seq
Id No. 459) CFTR13C-as 571 589 521 639 CCTCTTCGATGCCATTCAT (Seq Id
No. 460) CFTR13D-s 516 538 466 588
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCAATCCAATCAACTCTATACGAA
(Seq Id No. 461) CFTR13D-as 660 677 610 727 CTGATCACGCTGATGCGA (Seq
Id No. 462) CFTR13E-s 594 613 544 663
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGATGAGCCTTTAGAGAGAA (Seq
Id No. 463) CFTR13E-as 808 826 758 876 CCAGTTCAGTCAAGTTTGC (Seq Id
No. 464) CF13F-s2 666 679 616 729 CGCCCGCCGCGCCCCGCGCCCGCCCC-
GCCGCCCCCGCCCGCAGCGTGATCAGCA (Seq Id No. 465) CF13F-as2 960 977 910
1027 TTTGTTACATGCTACATA (Seq Id No. 466) CFTR13F-s 663 680 613 730
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCATCAGCGTGATCAGCA- C (Seq
Id No. 467) CFTR13F-as 960 985 910 1035 TAGTAAATTTTGTTACATGCTACATA
(Seq Id No. 468) HUMCFTRA14 CFTR14A-1-s 260 290 210 340
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTTCAT- ATATTAAAATAAAACC
(Seq Id No. 469) CFTR14A-1-as 398 418 348 468 TAATATATCGAAGGTATGTGT
(Seq Id No. 470) CFTR14A-2-s 372 393 322 443 GAGCATACCAGCAGTGACTACA
(Seq Id No. 471) CFTR14A-2-as 477 505 427 555
GCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGTAATACTTTACAATAGA-
ACATTCTTACC (Seq Id No. 472) CFTR14A-3-s 379 397 329 447
ACCAGCAGTGACTACATGGA (Seq Id No. 473) CFTR14A-3-as 542 569 492 619
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATATTTATGTGTGTGCATATATATGTAT
(Seq Id No. 474) HUMCFTRA15 CFTR14B-1-s 104 120 54 170
CGCCCGCCGCGCCCCGCGCCCGCCCCGCGCCCCGCCGGTGTACCTGATATTTGG (Seq Id No.
475) CFTR14B-1-as 247 262 197 312 CTCACTTTCCAAGGAG (Seq Id No. 476)
CF14B-3-s 240 254 190 304 GCTGTGGCTCCTTGG (Seq Id No. 477)
CF14B-3-as 490 507 440 557
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGA- CTACAGCCCTGAACTCC (Seq
Id No. 478) CFTR14B-2-s 220 238 170 288 GTGGCTGCTTCTTTGGTTG (Seq Id
No. 479) CFTR14B-2-as 305 333 255 383
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTACTAGTGGATTACAATATTTAAT
(Seq Id No. 480) HUMCFTRA16 CFTR15A-s 299 324 249 374
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCGCATGTATTGGAAATTCAGTAAGTAAC (Seq
Id No. 481) CFTR15A-as 519 542 469 592 TTCGACACTGTGATTAGAGTATGC
(Seq Id No. 482) 50 CFTR15B-s 463 478 413 528 GTGGGAGTAGCCGACA (Seq
Id No. 483) CFTR15B-as 651 667 601 717
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCAGGCCCTATTGATGGT (Seq Id
No. 484) CF15B-s2: 462 477 412 527 CGTGGGAGTAGCCGAC (Seq Id No.
485) CF15B-as2: 672 689 622 739 GGCCCGCCGCGCCCCGCGCCCGCCCCGCC-
GCCCCCGCCCGCATTAGAAAACCAACAA (Seq Id No. 486) HUMCFTRA17 CF16A-s5
226 241 176 291 CCCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCTGAATGC-
GTCTACTG (Seq Id No. 487) CF16A-as5 354 370 304 420
CATCCAAAATTGCTATA (Seq Id No. 488) CFTR16A-s 233 258 183 308
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCGTCTACTGTGATCCAAACTTAGTAT
(Seq Id No. 489) CFTR16A-as 387 410 337 460
CATACCTGGATGAAGTCAAATATG (Seq Id No. 490) CFTR16B-s 296 318 246 368
TTGAGGAATTTGTCATCTTGTAT (Seq Id No. 491) CFTR16B-as 456 478 406 528
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGAAAATCACATTTGCTTT- TGTTA
(Seq Id No. 492) HUMCFTRA18 CF17A-1-s 130 147 80 197
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCAAAGAAATAAATCACTGA (Seq Id
No. 493) CF17A-1-as6 243 258 193 308 GTAAAACTGCGACAAC (Seq Id No.
494) CFTR17A-2-s 187 212 137 262 CCAACATGTTTTCTTTGATCTTA- CAG (Seq
Id No. 495) CFTR17A-2-as 399 425 349 475
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGAGAATCTCAAATAGCTCTTATAGCTTT
(Seq Id No. 496) HUMCFTRA19 CFTR18A-1-s 35 56 -15 106
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTTAACCAATGACATTTGTGATA (Seq
Id No. 497) CFTR17B-1-As 189 209 139 259 GTGTCCATAGTCCTTTTAAGC (Seq
Id No. 498) CFTR17B-2-s 145 165 95 215
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATATTTCACAGGCAGGAGTCC (Seq
Id No. 499) CFTR17B-2-as 312 336 262 386 AAAATCATTTCTATTCTCATTTGGA
(Seq Id No. 500) CFTR17B-3-s 208 224 158 274 ACTTCGTGCCTTCGGAC (Seq
Id No. 501) CFTR17B-3-as 335 356 285 406
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCAGAATGAAGAAGATGACAAA (Seq
Id No. 502) CFTR17B-4-s 307 323 257 373 CTGGTTCCAAATGAGAA (Seq Id
No. 503) CFTR7B-4-as 444 462 394 512 CGCCCGCCGCGCCCCGCGCCCGCCC-
CGCCGCCCCCGCCCGTAACCTATAGAATGCAGCA (Seq Id No. 504) HUMCFTRA20
CFTR18A-s 239 258 189 308 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCG-
CCCGTTAATGTGATATGTGCCCTA (Seq Id No. 505) CFTR18A-as 431 451 381
501 AGATGATAAGACTTACCAAGC (Seq Id No. 506) CFTR18B-s 335 355 285
405 GAGAAGGAGAAGGAAGAGTTG (Seq Id No. 507) CFTR18B-as 490 512 440
562 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCTTCCTTCATGCTATTACTCA-
TAC (Seq Id No. 508) HUMCFTRA21 CFTR19A-s2 103 123 53 173
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGAAGTTATTTTTAGGAAGCAT (Seq
Id No. 509) CFTR19A-as 197 215 147 265 GAACTTAAAGACTCGGCTC (Seq Id
No. 510) CFTR19B-s 136 156 86 206 CGCCCGCCGCGCCCCGCGCCCGCCCC-
GCCGCCCCCGCCCGGAAATTGTCTGCCATTCTTAA (Seq Id No. 511) CFTR19b-as 259
278 209 328 GAGTTGGCCATTCTTGTATG (Seq Id No. 512) CF19C-s3 194 209
144 259 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGTGAGCC- GAGTCTTT
(Seq Id No. 513) CF19C-as2 379 394 329 444 ATGGCATTTCCACCTT (Seq Id
No. 514) CF19C-s2 169 189 119 239
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGTTATTTTTATTTCAGATGC (Seq
Id No. 515) CF19C-as2 380 398 330 448 TAATATGGCATTTCCACCT (Seq Id
No. 516) CF19D-s2 308 323 258 373 CGTGAAGAAAGATGAC (Seq Id No. 517)
CF19D-as2 513 531 463 581 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCG-
CCCCCGCCCGTAATGTTACAAATAGATTC (Seq Id No. 518) CFTR19D-s 304 324
254 374 CACACGTGAAGAAAGATCACA (Seq Id No. 519) CFTR19D-as 506 531
456 581 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTAATGTTAATAGATTCT-
GCTAAAC (Seq Id No. 520) dssp intronic: no alignment CF191-s2
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCTTGATTTCT- GGAGAC (Seq Id
No. 521) to AH006034 CF191-as2 CTAGCTGTAATTGCAT (Seq Id No. 522)
HUMCFTRA22 CFTR20-s 203 223 153 273
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCAATTATGTTTATGGCATGGT (Seq Id
No. 523) CFTR20-as 470 494 420 544 GAGTACAASTATCAAATAGCAGTAA (Seq
Id No. 524) new 20-s: 201 219 151 269
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCTGAATTATGTTTATGGCA (Seq Id
No. 525) new 20-as 435 452 385 502 CCTTTTTTCTGGCTAAGT (Seq Id No.
526) HUMCFTRA23 CFTR21A-s 162 182 112 232
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGATGGTAAGTACATGGGTGTT (Seq
Id No. 527) CFTR21A-as 333 353 283 403 ACTCCACTGTTCATAGGGATC (Seq
Id No. 528) CF 21A-s3: 254 273 204 323 CGCCCGCCGCGCCCCGCGCCCGCC-
CCGCCGCCCCCGCCCGAGTTATTCATACTTTCTTCT (Seq Id No. 529) CF 21A-as3:
376 391 326 441 AGCCTTACCTCATCTG (Seq Id No. 530) CF21B-s3 307 322
257 372 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTTTCTGGA- ACATTTAG
(Seq Id No. 531) CF21B-as3 443 460 393 510 GAATGATGTCAGCTATAT (Seq
Id No. 532) CFTR21B-s2 307 329 257 379
CGCCCGCCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTTTCTGGAACATTTAGAAAAAAC (Seq
Id No. 533) CFTR21B-as2 544 562 494 612 TCAGTTAGGGGTAGGTCCA (Seq Id
No. 534) HUMCFTRA24 CFTR22A-s2 356 373 306 423
CGCCCGCCCCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGAGCTGTCAAGGTTGTA (Seq Id
No. 535) CFTR22A-as2 551 569 501 619 CAGGAAACTGTTCTATCAC (Seq Id
No. 536) CFTR22B-s 474 497 424 547 CGCCCGCCGCGCCCCGCGCCCGCCC-
CGCCGCCCCCGCCCGGAATGTCAACTGCTTGAGTGTTTT (Seq Id No. 537) CFTR22B-as
707 733 657 783 AAGTAACAGAACATCTGAAACTCACAC (Seq Id No. 538)
CFTR22C-s2 570 686 620 736 CTTGCTGCTTGATGAAC (Seq Id No. 539)
CFTR22C-as 821 843 771 893 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCC-
CCGCCCGTGGGCAATTATTTCATATCTTGG (Seq Id No. 540) HUMCFTRA25 CF23A-s3
223 240 173 290 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTATCAAGG-
TAAATACAGA (Seq Id No. 541) CF23A-as3 353 369 303 419
GCTTCTATCCTGTGTTC (Seq Id No. 542) CF233-s2 256 276 206 326
GATATTATGTGTGGTATTTTC (Seq Id No. 543) CF235-as2 477 495 427 545
GCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCCTTGTACATTGTTGCA (Seq Id No.
544) CFTR23-s 218 244 168 294 CGCCCGCCGCGCCCCGCGCCCGCCCC-
GCCGCCCCCGCCCGCATATTATCAAGGTAAATACAGATCAT (Seq Id No. S45)
CFTR23-as 445 469 395 519 GGAACTATCACATGTGAGATTGTTA (Seq Id No.
546) HUMCFTRA26 CF24A-s2 107 122 57 172
CGCCCGCCGCGCCCCGCGCCCGCCCCG- CCGCCCCCGCCCGCCTTTGAGCCTGTGCC (Seq Id
No. 547) CF24A-as2 300 315 250 365 GCTTGAGTTCCGGTGG (Seq Id No.
548) CF24B-s 267 282 217 332 CATCAGCCCCTCCGAC (Seq Id No. 549)
CF24B-s2 482 498 432 548
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTTTCTGATGAAGGCAGAGGTA (Seq
Id No. 550) CFTR24A-s 82 99 32 149
CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCATTGCATTCTTTGACTT (Seq Id
No. 551) CFTR24A-as 280 296 23 346 AAGAGCTTCACCCTGTC (Seq Id No.
552) CFTR24B-s 213 229 163 279 GCAGTACGATTCCATCC (Seq Id No. 553)
CFTR24B-as 489 504 439 554 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCC-
CCGCCCGCCTTGTTTTCTGACGC (Seq Id No. 554)
[0137]
6TABLE C PCR Plate Set Up Position 1 2 3 4 5 6 7 8 9 10 11 12 Plate
49 Fragment 16B 6B2 17B4 14A1 6A2 11A Multi G 3A 3B1 3B2 5A2 10B2
5F Plate 52 Fragment 17B1 14A3 3A 11B 7B 21B 22C 22A Multi G 3A3
4B3 5D1 6A2 8A1 7B 8A 7B Plate 54 Fragment 5A 3B 19A 4B 2A 19C 14B1
22B 17B2 6B1 Multi G 4A 4B2 5B2 6A3 7B 8B2 8B4 10A1 10A2 4C Plate
59 A Fragment 8B 13F 10 18B 19B 18A 14B2 Multi G 5B 5C 5E 5F 6A 7A
8A Plate 59 B Fragment 15B 13D 24B 24A 13E 1A 1B Multi G 8B 9A 11A
11A 12A 13A 13A Plate 62.4 A Fragment 21A 8A 13A 7D 23 14A2 13B 13C
15A 9C 2B 5B Multi G 1A 2A 4A 4B 5A 5D 6A 7A 7A 7B 1B 4C Plate 62.4
B Fragment 12 6A3 17A2 19D 20 4A 17B3 7C 6A1 16A 7A 61 total
fragments
[0138]
7TABLE D Group TTGE Start TTGE End Gels/Group Dcodes 1 44.5 48.0 2
1 2 48.0 50.5 1 1 3 48.0 51.0 2 1 4 49.0 52.5 3 2 5 50.5 53.5 6 3 6
52.5 55.5 1 1 7 53.5 56.5 2 1 8 55.0 58.0 2 1 9 56.0 58.5 2 1 10
57.0 59.5 2 1 11 58.5 62.0 1 1 12 58.5 62.0 1 1 13 62.0 64.0 1 1
Totals: 26 16
[0139]
8TABLE E PCR Length.sub.-- Min Max Min Max Min Max Gene Fragment
Temp Rating App Tm Length GC PCR PCR Melt Melt Actual Actual Group
Position CFTR 21A 55 81 52 192 242 45 65 60 62 46 48 1 A1 CFTR 2B
60 60 57 199 249 45 56.7 61 62 47 48 1 B1 CFTR 8A 55 60 48 152 202
50 62 62 64 48 50 2 A1 CFTR 16A 55 80 56 178 228 45 65 62 64 48 50
2 A2 CFTR 7A 55 78 63 118 168 50 65 62 64 48 50 2 A3 CFTR 12 50 80
55 238 288 45 65 63 65 49 51 3 A1 CFTR 18B 50 78 54 183 233 45 65
63 65 49 51 3 A2 CFTR 17B-1 53 83 51 175 225 45 59.6 63 65 49 51 3
A3 CFTR 6B-2 50 86 36 159 209 48 59 63 65 49 51 3 B1 CFTR 17B-4 50
78 45 156 206 45 56.7 63 65 49 51 3 B2 CFTR 13A 50 73 38.5 209 259
45 65 63.5 66.5 49.5 52.5 4 A1 CFTR 5A 53 77 52 139 189 45 59.6 65
66 51 52 4 A2 CFTR 7D 62 64 56 161 211 45 65 64 66 50 52 4 B1 CFTR
3B 55 87 57 177 227 45 65 65 66 51 52 4 B2 CFTR 14A-3 53 63 53 192
242 45 65 65 66 51 52 4 B3 CFTR 6B-1 -- 88 33 148 198 46.5 65 64 66
50 52 4 C1 CFTR 5B -- 92 44 163 213 59.6 65 64 66 50 52 4 C2 CFTR
23 53 64 55 252 302 65 67 51 53 5 A1 CFTR 14A-1 55 50 45 65 66 68
52 54 5 A2 CFTR 9A 60 46 171 221 45 63.4 66 67 52 53 5 CFTR 8B 53
57 49 227 277 45 65 66 67 51 53 5 B1 CFTR 19A 50 84 49 166 216 45
65 66 67 51 53 5 B2 CFTR 17A-1 55 66 54 181 231 50.6 65 66 67 52 53
5 CFTR 11A 50 67 52 144 194 48.2 65 66 67 51 53 5 F1 CFTR 18B 62 68
52 178 228 45 64.6 66 68 52 54 5 F2 CFTR 13F 50 66 42 312 382 45 65
67 68 53 54 5 C1 CFTR 8A-3 50 70 56 163 233 53.4 63 66 68 52 54 5
C2 CFTR 3A 50 72 44 230 280 45 56.7 66 68 52 54 5 D1 CFTR 14A-2 50
80 55 134 184 53.4 65 67 68 53 54 5 D2 CFTR 9B 60 68 53 167 207 45
65 65 68 51 53 5 CFTR 10 60 92 50 356 406 20.5 65 65 68 51 54 5 E2
CFTR 13B 55 64 55 440 490 45 61.8 66.5 68.5 52.5 54.5 6 A1 CFTR 11B
45 74 43 180 230 59.6 65 67 69 53 55 6 A2 CFTR 4B 55 92 43 194 244
45 59 67 69 53 55 6 A3 CFTR 19B 60 48 53 143 193 50 65 67 69 53 55
6 A4 CFTR 9D 60 53 56 132 182 45 63.4 67 69 53 56 6 CFTR 13C 45 81
55 300 350 45 61.8 68 70 54 56 7 A1 CFTR 18A 62 92 49 213 263 50.5
63.4 68 70 54 56 7 A2 CFTR 17A-2 55 59 58 239 289 45 65 68 70 54 56
7 A3 CFTR 15A 50 82 56 244 294 45 65 69 70 55 56 7 A4 CFTR 9C 60 65
56 200 250 63.4 65 69 70 55 56 7 B1 CFTR 22A 55 76 53 217 267 45 65
67.5 70 53.5 56 7 B2 CFTR 2A 53 80 52 166 216 45 61.8 68 70 54 56 7
B3 CFTR 21B 76 53 256 306 45 65 68 70 54 56 7 B4 CFTR 7B 55 93 61
116 166 45 65 70 71 56 57 8 A1 CFTR 14B-2 50 69 56 114 164 45 65 69
71 56 57 8 A2 CFTR 22C 50 76 47 167 217 45 65 69 71 55 57 8 A3 CFTR
19D 50 50 45 65 69.5 71.5 55.5 57.5 8 A4 CFTR 20 50 71 53 228 278
45 65 69 71 55 57 8 B1 CFTR 19C 53 84 50 226 276 45 65 69 71 55 57
8 B2 CFTR 15B 45 82 49 205 255 45 53.4 69 71 55 57 8 B3 CFTR 14B-1
45 75 40 159 209 45 65 70 71 56 57 8 B4 CFTR 14A 50 79 55 219 269
45 63.4 70 72 56 58 9 A1 CFTR 13D 50 78 55 162 212 48.2 56.7 70 73
56 59 9 A2 CFTR 17B-3 45 89 54 149 199 45 56.7 71 72 57 58 9 B1
CFTR 22B 55 66 58 260 310 45 65 71 73 57 59 10 A1 CFTR 17B-2 45 80
56 192 242 45 59.6 71 73 57 59 10 A2 CFTR 7C 55 85 55 253 303 45 65
71 73 57 59 10 B1 CFTR 6A-2 50 93 49 127 177 45 65 71 73 57 59 10
B2 CFTR 6A-1 50 86 46 292 342 45 65 73 76 59 61 11 A1 CFTR 24B 50
88 63 183 233 73 74 59 60 11 A2 CFTR 24A 50 81 46 215 265 73 75 59
61 11 A3 CFTR 13E 50 50 45 65 73 76.5 59 62.5 12 A1 CFTR 1A 60 84
62 180 230 56.7 65 76 78 62 64 13 A1 CFTR 1B 60 85 60 165 215 56.7
64.6 76 77 62 63 13 A2
[0140]
Sequence CWU 1
1
554 1 58 DNA Artificial Sequence Diagnostic Oligonucleotide 1
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg ggatgataat tggaggca 58
2 20 DNA Artificial Sequence Diagnostic Oligonucleotide 2
taggagaagt gtgaataaag 20 3 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 3 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ctgttctgtg atattatgtg 60 4 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 4 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tttgcttctc cagttgaaca 60 5 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 5 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ttgaagtgtc caccaaaatg 60 6 61 DNA Artificial Sequence Diagnostic
Oligonucleotide 6 cgcccgccgc gccccgcgcc cgcccctgcc gcccccgccc
ggtactatcc ccaagtaacc 60 t 61 7 67 DNA Artificial Sequence
Diagnostic Oligonucleotide 7 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg tacagtggat atagaaagga 60 caatttt 67 8 25 DNA Artificial
Sequence Diagnostic Oligonucleotide 8 cagattctct acttcatagc catag
25 9 62 DNA Artificial Sequence Diagnostic Oligonucleotide 9
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg ctatttatgg ttttgcttgt
60 gg 62 10 60 DNA Artificial Sequence Diagnostic Oligonucleotide
10 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg gctcagtata
actgaggctg 60 11 59 DNA Artificial Sequence Diagnostic
Oligonucleotide 11 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
caaaagttga tggcagagt 59 12 18 DNA Artificial Sequence Diagnostic
Oligonucleotide 12 tgtcaggcca attacaga 18 13 58 DNA Artificial
Sequence Diagnostic Oligonucleotide 13 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg ggggtgagga attttgaa 58 14 59 DNA Artificial
Sequence Diagnostic Oligonucleotide 14 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg atacccttat tccctgtgg 59 15 20 DNA Artificial
Sequence Diagnostic Oligonucleotide 15 cactggttgg gctagtatgt 20 16
58 DNA Artificial Sequence Diagnostic Oligonucleotide 16 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg cccagtgttg agcctttg 58 17 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 17 gctcccagta
gggtcagcat c 21 18 20 DNA Artificial Sequence Diagnostic
Oligonucleotide 18 atggccaagt actaggttgg 20 19 20 DNA Artificial
Sequence Diagnostic Oligonucleotide 19 ctaaccgatt gaatatggag 20 20
60 DNA Artificial Sequence Diagnostic Oligonucleotide 20 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg atactttgtt acttgtctga 60 21 20 DNA
Artificial Sequence Diagnostic Oligonucleotide 21 gttatcaaga
attacaaggg 20 22 20 DNA Artificial Sequence Diagnostic
Oligonucleotide 22 cgatcagacc ctacaggaca 20 23 27 DNA Artificial
Sequence Diagnostic Oligonucleotide 23 gatacccaat ttcataaata
gcattca 27 24 22 DNA Artificial Sequence Diagnostic Oligonucleotide
24 catagaatga caggacaata gg 22 25 27 DNA Artificial Sequence
Diagnostic Oligonucleotide 25 tgcttatttc atctcaatcc tacgctt 27 26
67 DNA Artificial Sequence Diagnostic Oligonucleotide 26 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg aatattcatt ttaaagatcc 60 aagatat
67 27 25 DNA Artificial Sequence Diagnostic Oligonucleotide 27
taaggggaca tacactgaga atgaa 25 28 20 DNA Artificial Sequence
Diagnostic Oligonucleotide 28 ctctgagtca gttaaacagt 20 29 18 DNA
Artificial Sequence Diagnostic Oligonucleotide 29 atgactttat
ggcaggga 18 30 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 30 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tacggaaatg gtataggaaa 60 31 19 DNA Artificial Sequence Diagnostic
Oligonucleotide 31 ggtgaggggt gtaatggtt 19 32 21 DNA Artificial
Sequence Diagnostic Oligonucleotide 32 atggctctat gtcatcttgt c 21
33 58 DNA Artificial Sequence Diagnostic Oligonucleotide 33
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg taggttgagg gttgggac 58
34 21 DNA Artificial Sequence Diagnostic Oligonucleotide 34
cctcgtggtg tagagtgatg t 21 35 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 35 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gagcccagga gcccagaaat 60 36 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 36 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gagggccata gactatagca 60 37 20 DNA Artificial Sequence Diagnostic
Oligonucleotide 37 tttctgtccc tgctctggtc 20 38 60 DNA Artificial
Sequence Diagnostic Oligonucleotide 38 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg tcccacgagc tccaattcca 60 39 58 DNA Artificial
Sequence Diagnostic Oligonucleotide 39 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg ggtgaggtct gggaagtg 58 40 19 DNA Artificial
Sequence Diagnostic Oligonucleotide 40 tgcctccttg agtatctgc 19 41
60 DNA Artificial Sequence Diagnostic Oligonucleotide 41 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg cagccaagcc ctacctctcg 60 42 20 DNA
Artificial Sequence Diagnostic Oligonucleotide 42 cttcatcacc
ccctccctgc 20 43 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 43 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
cttgatgact tcctatccat 60 44 21 DNA Artificial Sequence Diagnostic
Oligonucleotide 44 aacctccatc cagtgcctag c 21 45 1577 DNA
Artificial Sequence Diagnostic Oligonucleotide 45 ccacccttgg
agttcactca cctaaacctc aaactaataa agcttggttc ttttctccga 60
cacgcaaagg aagcgctaag gtaaatgcat cagacccaca ctgccgcgga acttttcggc
120 tctctaaggc tgtattttga tatacgaaag gcacattttc cttccctttt
caaaatgcac 180 cttgcaaacg taacagggac ccgactagga tcatcgggaa
aaggaggagg aggaggaagg 240 caggctccgg ggaagctggt ggcagcgggt
cctgggtctg gcggaccctg acgcgaagga 300 gggtctagga agctctccgg
ggagccgttc tcccgccggt ggcttcttct gtcctccagc 360 gttgccaact
ggacctaaag agaggccgcg actgtcgccc acctgcggga tgggcctggt 420
gctgggcggt aaggacacgg acctggaagg agcgcgcgcg agggagggag gctgggagtc
480 agaatcggga aagggaggtg cggggcggcg agggagcgaa ggaggagagg
aggaaggagc 540 gggaggggtg ctggcggggg tgcgtagtgg gtggagaaag
ccgctagagc aaatttgggg 600 ccggaccagg cagcactcgg cttttaacct
gggcagtgaa ggcgggggaa agagcaaaag 660 gaaggggtgg tgtgcggagt
aggggtgggt ggggggaatt ggaagccaaa tgacatcaca 720 gcaggtcaga
gaaaaagggt tgagcggcag gcacccagag tagtaggtct ttggcattag 780
gagcttgagc ccagacggcc ctagcaggga ccccagcgcc cagagaccat gcagaggtcg
840 cctctggaaa aggccagcgt tgtctccaaa ctttttttca ggtgagaagg
tggccaaccg 900 agcttcggaa agacacgtgc ccacgaaaga ggagggcgtg
tgtatgggtt gggtttgggg 960 taaaggaata agcagttttt aaaaagatgc
gctatcattc attgttttga aagaaaatgt 1020 gggtattgta gaataaaaca
gaaagcatta agaagagatg gaagaatgaa ctgaagctga 1080 ttgaatagag
agccacatct acttgcaact gaaaagttag aatctcaaga ctcaagtacg 1140
ctactatgca cttgttttat ttcatttttc taagaaacta aaaatacttg ttaataagta
1200 cctagtatgg tttattggtt ttcccccttc atgccttgga cacttgattg
tcttcttggc 1260 acatacaggt gccatgcctg catatagtaa gtgctcagaa
aacatttctt gactgaattc 1320 agccaacaaa aattttgggg taggtagaaa
atatatgctt aaagtattta ttgttatgag 1380 actggatata tctagtattt
gtcacaggta aatgattctt caaaaattga aagcaaattt 1440 gttgaaatat
ttattttgaa aaaagttact tcacaagcta taaattttaa aagccatagg 1500
aatagatacc gaagttatat ccaactgaca tttaataaat tgtattcata gcctaatgtg
1560 atgagccaca gaagctt 1577 46 20 DNA Artificial Sequence
Diagnostic Oligonucleotide 46 taatggatca tgggccatgt 20 47 60 DNA
Artificial Sequence Diagnostic Oligonucleotide 47 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg aagagacatg gacaccaaat 60 48 62 DNA
Artificial Sequence Diagnostic Oligonucleotide 48 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ggaagccaaa tgacatcaca 60 gc 62 49
24 DNA Artificial Sequence Diagnostic Oligonucleotide 49 tgaaaaaaag
tttggagaca acgc 24 50 17 DNA Artificial Sequence Diagnostic
Oligonucleotide 50 cccagcgccc agagacc 17 51 62 DNA Artificial
Sequence Diagnostic Oligonucleotide 51 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg actgcttatt cctttacccc 60 aa 62 52 63 DNA
Artificial Sequence Diagnostic Oligonucleotide 52 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ccagaaaagt tgaatagtat 60 cag 63 53
19 DNA Artificial Sequence Diagnostic Oligonucleotide 53 agattgtcag
cagaatcaa 19 54 17 DNA Artificial Sequence Diagnostic
Oligonucleotide 54 ataccaaatc ccttctg 17 55 59 DNA Artificial
Sequence Diagnostic Oligonucleotide 55 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg tgctttctct tctctaaat 59 56 56 DNA Artificial
Sequence Diagnostic Oligonucleotide 56 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg tggtgttgta tggtct 56 57 17 DNA Artificial
Sequence Diagnostic Oligonucleotide 57 aacataaatc tccagaa 17 58 22
DNA Artificial Sequence Diagnostic Oligonucleotide 58 gctggcttca
aagaaaaatc cc 22 59 62 DNA Artificial Sequence Diagnostic
Oligonucleotide 59 gcccgccgcg ccccgcgccc gccccgccgc ccccgcccgc
accagatttc gtagtctttt 60 ca 62 60 61 DNA Artificial Sequence
Diagnostic Oligonucleotide 60 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg aatttctctg tttttcccct 60 t 61 61 22 DNA Artificial
Sequence Diagnostic Oligonucleotide 61 agctattctc atctgcattc ca 22
62 17 DNA Artificial Sequence Diagnostic Oligonucleotide 62
gacactgctc ctacacc 17 63 57 DNA Artificial Sequence Diagnostic
Oligonucleotide 63 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tcagcattta tccctta 57 64 66 DNA Artificial Sequence Diagnostic
Oligonucleotide 64 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ataatatatt tgtattttgt 60 ttgttg 66 65 20 DNA Artificial Sequence
Diagnostic Oligonucleotide 65 aatttgttca ggttgttgga 20 66 18 DNA
Artificial Sequence Diagnostic Oligonucleotide 66 agctgtcaag
ccgtgttc 18 67 59 DNA Artificial Sequence Diagnostic
Oligonucleotide 67 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
atctgaccca ggaaaactc 59 68 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 68 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ttgttagttt ctaggggtgg 60 69 21 DNA Artificial Sequence Diagnostic
Oligonucleotide 69 aaggactatc aggaaaccaa g 21 70 20 DNA Artificial
Sequence Diagnostic Oligonucleotide 70 gctaatctgg gagttgttac 20 71
61 DNA Artificial Sequence Diagnostic Oligonucleotide 71 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg agttatgaaa ataggttgca 60 c 61 72
58 DNA Artificial Sequence Diagnostic Oligonucleotide 72 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg gggagaatga tgatgaag 58 73 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 73 acactgaaga
tcactgttct a 21 74 63 DNA Artificial Sequence Diagnostic
Oligonucleotide 74 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ccttgagcag ttcttaatag 60 ata 63 75 21 DNA Artificial Sequence
Diagnostic Oligonucleotide 75 atgccttaac agattggata t 21 76 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 76 gaaaatatcc
aatctgttaa g 21 77 58 DNA Artificial Sequence Diagnostic
Oligonucleotide 77 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tgaggtggaa gtctacca 58 78 59 DNA Artificial Sequence Diagnostic
Oligonucleotide 78 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
agaccatgct cagatcttc 59 79 24 DNA Artificial Sequence Diagnostic
Oligonucleotide 79 gctgccttcc gagtcagttt cagt 24 80 56 DNA
Artificial Sequence Diagnostic Oligonucleotide 80 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg actgaaactg actcgg 56 81 26 DNA
Artificial Sequence Diagnostic Oligonucleotide 81 atggtacatt
acctgtattt tgttta 26 82 26 DNA Artificial Sequence Diagnostic
Oligonucleotide 82 ctgtacaaac atggtatgac tctctt 26 83 65 DNA
Artificial Sequence Diagnostic Oligonucleotide 83 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg gtgaaggaaa tttctttttc 60 tatct 65
84 62 DNA Artificial Sequence Diagnostic Oligonucleotide 84
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg gcacaatgag agtataaagt
60 ag 62 85 21 DNA Artificial Sequence Diagnostic Oligonucleotide
85 ccatcactac ttctgtagtc g 21 86 27 DNA Artificial Sequence
Diagnostic Oligonucleotide 86 ctctctttta taaataggat ttcttac 27 87
65 DNA Artificial Sequence Diagnostic Oligonucleotide 87 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ttccagttct accagttata 60 tcatc 65
88 65 DNA Artificial Sequence Diagnostic Oligonucleotide 88
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg acaatagaaa aacttctaat
60 ggtga 65 89 22 DNA Artificial Sequence Diagnostic
Oligonucleotide 89 aaaaaagaga catggacacc aa 22 90 18 DNA Artificial
Sequence Diagnostic Oligonucleotide 90 cctgagcgtg atttgata 18 91 60
DNA Artificial Sequence Diagnostic Oligonucleotide 91 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg atgtagacta accgattgaa 60 92 16 DNA
Artificial Sequence Diagnostic Oligonucleotide 92 gggagaactg gagcct
16 93 58 DNA Artificial Sequence Diagnostic Oligonucleotide 93
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg aaccgattga atatggag 58
94 61 DNA Artificial Sequence Diagnostic Oligonucleotide 94
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg gatatatgat tacattagaa
60 g 61 95 17 DNA Artificial Sequence Diagnostic Oligonucleotide 95
accttctcca agaacta 17 96 18 DNA Artificial Sequence Diagnostic
Oligonucleotide 96 ataggacatc tccaagtt 18 97 59 DNA Artificial
Sequence Diagnostic Oligonucleotide 97 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg gcaatagaga aatgtctgt 59 98 63 DNA Artificial
Sequence Diagnostic Oligonucleotide 98 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg gtgaactgtt taaggcaaat 60 cat 63 99 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 99 tgatgggaca
gtctgtcttt c 21 100 25 DNA Artificial Sequence Diagnostic
Oligonucleotide 100 aatacgagac atattgcaat aaagt 25 101 62 DNA
Artificial Sequence Diagnostic Oligonucleotide 101 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ctggctgtag attttggagt 60 tc 62 102
17 DNA Artificial Sequence Diagnostic Oligonucleotide 102
aggtagcagc tattttt 17 103 57 DNA Artificial Sequence Diagnostic
Oligonucleotide 103 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ggacagcctt ctctcta 57 104 61 DNA Artificial Sequence Diagnostic
Oligonucleotide 104 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
atgggacatt ttcagaactc 60 c 61 105 19 DNA Artificial Sequence
Diagnostic Oligonucleotide 105 cctcttcgat gccattcat 19 106 60 DNA
Artificial Sequence Diagnostic Oligonucleotide 106 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg tgatgagcct ttagagagaa 60 107 19
DNA Artificial Sequence Diagnostic Oligonucleotide 107 ccagttcagt
caagtttgc 19 108 54 DNA Artificial Sequence Diagnostic
Oligonucleotide 108 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
cagcgtgatc agca 54 109 18 DNA Artificial Sequence Diagnostic
Oligonucleotide 109 tttgttacat gctacata 18 110 62 DNA Artificial
Sequence Diagnostic Oligonucleotide 110 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg ttcatatatt aaaaataaaa 60 cc 62 111 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 111 taatatatcg
aaggtatgtg t 21 112 22 DNA Artificial Sequence Diagnostic
Oligonucleotide 112 gagcatacca gcagtgacta ca 22 113 69 DNA
Artificial Sequence Diagnostic Oligonucleotide 113 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg gtaatacttt acaatagaac 60 attcttacc
69 114 20 DNA Artificial Sequence Diagnostic Oligonucleotide 114
accagcagtg actacatgga 20 115 68 DNA Artificial Sequence Diagnostic
Oligonucleotide 115 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
atatttatgt gtgtgcatat 60 atatgtat 68 116 57 DNA Artificial Sequence
Diagnostic Oligonucleotide 116 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg gtgtaccttg atattgg 57 117 16 DNA Artificial Sequence
Diagnostic Oligonucleotide 117 ctcactttcc aaggag 16 118 15 DNA
Artificial Sequence Diagnostic Oligonucleotide 118 gctgtggctc cttgg
15 119 58 DNA Artificial Sequence Diagnostic Oligonucleotide 119
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg actacagccc tgaactcc 58
120 66 DNA Artificial Sequence Diagnostic Oligonucleotide 120
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg catgtattgg aaattcagta
60 agtaac 66 121 24 DNA Artificial Sequence Diagnostic
Oligonucleotide 121 ttcgacactg tgattagagt atgc 24 122 16 DNA
Artificial Sequence Diagnostic Oligonucleotide 122 cgtgggagta
gccgac 16 123 58 DNA Artificial Sequence Diagnostic Oligonucleotide
123 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg cattagaaaa ccaacaaa
58 124 56 DNA Artificial Sequence Diagnostic Oligonucleotide 124
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg ctgaatgcgt ctactg 56
125 17 DNA Artificial Sequence Diagnostic Oligonucleotide 125
catccaaaat tgctata 17 126 23 DNA Artificial Sequence Diagnostic
Oligonucleotide 126 ttgaggaatt tgtcatcttg tat 23 127 63 DNA
Artificial Sequence Diagnostic Oligonucleotide 127 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg caaaatcaca tttgcttttc 60 tta 63
128 58 DNA Artificial Sequence Diagnostic Oligonucleotide 128
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg aaagaaataa atcactga 58
129 16 DNA Artificial Sequence Diagnostic Oligonucleotide 129
gtaaaactgc gacaac 16 130 26 DNA Artificial Sequence Diagnostic
Oligonucleotide 130 ccaacatgtt ttctttgatc ttacag 26 131 67 DNA
Artificial Sequence Diagnostic Oligonucleotide 131 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg agaatctcaa atagctctta 60 tagcttt
67 132 62 DNA Artificial Sequence Diagnostic Oligonucleotide 132
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg ttaaccaatg acatttgtga
60 ta 62 133 21 DNA Artificial Sequence Diagnostic Oligonucleotide
133 gtgtccatag tccttttaag c 21 134 61 DNA Artificial Sequence
Diagnostic Oligonucleotide 134 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg atatttcaca ggcaggagtc 60 c 61 135 25 DNA Artificial
Sequence Diagnostic Oligonucleotide 135 aaaatcattt ctattctcat ttgga
25 136 17 DNA Artificial Sequence Diagnostic Oligonucleotide 136
acttcgtgcc ttcggac 17 137 62 DNA Artificial Sequence Diagnostic
Oligonucleotide 137 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
cagcaatgaa gaagatgaca 60 aa 62 138 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 138 ctggttccaa atgagaa 17 139 59 DNA
Artificial Sequence Diagnostic Oligonucleotide 139 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg taacctatag aatgcagca 59 140 60 DNA
Artificial Sequence Diagnostic Oligonucleotide 140 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ttaatgtgat atgtgcccta 60 141 21
DNA Artificial Sequence Diagnostic Oligonucleotide 141 agatgataag
acttaccaag c 21 142 21 DNA Artificial Sequence Diagnostic
Oligonucleotide 142 gagaaggaga aggaagagtt g 21 143 63 DNA
Artificial Sequence Diagnostic Oligonucleotide 143 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg cttcctcatg ctattactca 60 tac 63
144 61 DNA Artificial Sequence Diagnostic Oligonucleotide 144
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg aagttatttt ttaggaagca
60 t 61 145 19 DNA Artificial Sequence Diagnostic Oligonucleotide
145 gaacttaaag actcggctc 19 146 61 DNA Artificial Sequence
Diagnostic Oligonucleotide 146 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg gaaattgtct gccattctta 60 a 61 147 20 DNA Artificial
Sequence Diagnostic Oligonucleotide 147 gagttggcca ttcttgtatg 20
148 56 DNA Artificial Sequence Diagnostic Oligonucleotide 148
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg tgtgagccga gtcttt 56
149 16 DNA Artificial Sequence Diagnostic Oligonucleotide 149
atggcatttc cacctt 16 150 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 150 cgtgaagaaa gatgac 16 151 59 DNA Artificial
Sequence Diagnostic Oligonucleotide 151 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg taatgttaca aatagattc 59 152 56 DNA Artificial
Sequence Diagnostic Oligonucleotide 152 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg cttgatttct ggagac 56 153 16 DNA Artificial
Sequence Diagnostic Oligonucleotide 153 ctagctgtaa ttgcat 16 154 59
DNA Artificial Sequence Diagnostic Oligonucleotide 154 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ctgaattatg tttatggca 59 155 18 DNA
Artificial Sequence Diagnostic Oligonucleotide 155 ccttttttct
ggctaagt 18 156 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 156 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
agttattcat actttcttct 60 157 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 157 agccttacct catctg 16 158 56 DNA Artificial
Sequence Diagnostic Oligonucleotide 158 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg tttctggaac atttag 56 159 18 DNA Artificial
Sequence Diagnostic Oligonucleotide 159 gaatgatgtc agctatat 18 160
58 DNA Artificial Sequence Diagnostic Oligonucleotide 160
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg tgagctgtca aggttgta 58
161 19 DNA Artificial Sequence Diagnostic Oligonucleotide 161
caggaaactg ttctatcac 19 162 64 DNA Artificial Sequence Diagnostic
Oligonucleotide 162 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gaatgtcaac tgcttgagtg 60 tttt 64 163 27 DNA Artificial Sequence
Diagnostic Oligonucleotide 163 aagtaacaga acatctgaaa ctcacac 27 164
17 DNA Artificial Sequence Diagnostic Oligonucleotide 164
cttgctgctt gatgaac 17 165 63 DNA Artificial Sequence Diagnostic
Oligonucleotide 165 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tgggcaatta tttcatatct 60 tgg 63 166 58 DNA Artificial Sequence
Diagnostic Oligonucleotide 166 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg tatcaaggta aatacaga 58 167 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 167 gcttctatcc tgtgttc 17 168 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 168 gatattatgt
gtggtatttt c 21 169 59 DNA Artificial Sequence Diagnostic
Oligonucleotide 169 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gaacttgtac attgttgca 59 170 56 DNA Artificial Sequence Diagnostic
Oligonucleotide 170 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
cctttgagcc tgtcgg 56 171 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 171 gcttgagttc cggtgg 16 172 16 DNA Artificial
Sequence Diagnostic Oligonucleotide 172 catcagcccc tccgac 16 173 57
DNA Artificial Sequence Diagnostic Oligonucleotide 173 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg tttctgaggc agaggta 57 174 659 DNA
Artificial Sequence Diagnostic Oligonucleotide 174 gggaggggtg
ctggcggggt gcgtagtggg tggagaaagc cgctagagca aatttggggc 60
cggaccaggc agcactcggc ttttaacctg ggcagtgaag gcgggggaaa gagcaaaagg
120 aaggggtggt gtgcggagta ggggtgggtg gggggaattg gaagccaaat
gacatcacag 180 caggtcagag aaaaagggtt gagcggcagg cacccagagt
agtaggtctt tggcattagg 240 agcttgagcc cagacggccc tagcagggac
cccagcgccc agagaccatg cagaggtcgc 300 ctctggaaaa ggccagcgtt
gtctccaaac tttttttcag gtgagaaggt ggccaaccga 360 gcttcggaaa
gacacgtgcc cacgaaagag gagggcgtgt gtatgggttg ggtttggggt 420
aaaggaataa gcagttttta aaaagatgcg ctatcattca ttgttttgaa agaaaatgtg
480 ggtattgtag aataaaacag aaagcattaa gaagagatgg aagaatgaac
tgaagctgat 540 tgaatagaga gccacatcta cttgcaactg aaaagttaga
atctcaagac tcaagtacgc 600 tactatgcac ttgttttatt tcatttttct
aagaaactaa aaatacttgt taataagta 659 175 62 DNA Artificial Sequence
Diagnostic Oligonucleotide 175 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg ggaagccaaa tgacatcaca 60 gc 62 176 24 DNA Artificial
Sequence Diagnostic Oligonucleotide 176 tgaaaaaaag tttggagaca acgc
24 177 17 DNA Artificial Sequence Diagnostic Oligonucleotide 177
cccagcgccc agagacc 17 178 62 DNA Artificial Sequence Diagnostic
Oligonucleotide 178 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
actgcttatt cctttacccc 60 aa 62 179 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 179 gggtggtgtg cggagta 17 180 20 DNA
Artificial Sequence Diagnostic Oligonucleotide 180 caaaacaatg
aatgatagcg 20 181 630 DNA Artificial Sequence Diagnostic
Oligonucleotide 181 aaaccatact attattccct cccaatccct ttgacaaagt
gacagtcaca ttagttcaga 60 gatattgatg ttttatacag gtgtagcctg
taagagatga agcctggtat ttatagaaat 120 tgacttattt tattctcata
tttacatgtg cataattttc catatgccag aaaagttgaa 180 tagtatcaga
ttccaaatct gtatggagac caaatcaagt gaatatctgt tcctcctctc 240
tttattttag ctggaccaga ccaattttga ggaaaggata cagacagcgc ctggaattgt
300 cagacatata ccaaatccct tctgttgatt ctgctgacaa tctatctgaa
aaattggaaa 360 ggtatgttca tgtacattgt ttagttgaag agagaaattc
atattattaa ttatttagag 420 aagagaaagc aaacatatta taagtttaat
tcttatattt aaaaatagga gccaagtatg 480 gtggctaatg cctgtaatcc
caactatttg ggaggccaag atgagaggat tgcttgagac 540 caggagtttg
ataccagcct gggcaacata gcaagatgtt atctctacac aaaataaaaa 600
gttagctggg aatggtagtg catgcttgta 630 182 63 DNA Artificial Sequence
Diagnostic Oligonucleotide 182 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg ccagaaaagt tgaatagtat 60 cag 63 183 19 DNA Artificial
Sequence Diagnostic Oligonucleotide 183 agattgtcag cagaatcaa 19 184
19 DNA Artificial Sequence Diagnostic Oligonucleotide 184
gacagtcaca ttagttcag 19 185 17 DNA Artificial Sequence Diagnostic
Oligonucleotide 185 tgtttgcttt ctcttct 17 186 17 DNA Artificial
Sequence Diagnostic Oligonucleotide 186 ataccaaatc ccttctg 17 187
59 DNA Artificial Sequence Diagnostic Oligonucleotide 187
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg tgctttctct tctctaaat 59
188 454 DNA Artificial Sequence Diagnostic Oligonucleotide 188
aggaatctgc cagatatctg gctgagtgtt tggtgttgta tggtctccat gagattttgt
60 ctctataata cttgggttaa tctccttgga tatacttgtg tgaatcaaac
tatgttaagg 120 gaaataggac aactaaaata tttgcacatg caacttattg
gtcccacttt ttattctttt 180 gcagagaatg ggatagagag ctggcttcaa
agaaaaatcc taaactcatt aatgcccttc 240 ggcgatgttt tttctggaga
tttatgttct atggaatctt tttatattta ggggtaagga 300 tctcatttgt
acattcatta tgtatcacat aactatatgc atttttgtga ttatgaaaag 360
actacgaaat ctggtgaata ggtgtaaaaa tataaaggat gaatccaact ccaaacacta
420 agaaaccacc taaaactcta gtaaggataa gtaa 454 189 56 DNA Artificial
Sequence Diagnostic Oligonucleotide 189 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg tggtgttgta tggtct 56 190 17 DNA Artificial
Sequence Diagnostic Oligonucleotide 190 aacataaatc tccagaa 17 191
21 DNA Artificial Sequence Diagnostic Oligonucleotide 191
gctggcttca aagaaaaatc c 21 192 63 DNA Artificial Sequence
Diagnostic Oligonucleotide 192 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg caccagattt cgtagtcttt 60 tca 63 193 17 DNA Artificial
Sequence Diagnostic Oligonucleotide 193 tggtgttgta tggtctc 17 194
19 DNA Artificial Sequence Diagnostic Oligonucleotide 194
ttaggtggtt ttcttagtg 19 195 664 DNA Artificial Sequence Diagnostic
Oligonucleotide 195 ccactattca ctgtttaact taaaatacct catatgtaaa
cttgtctccc actgttgcta 60 taacaaatcc caagtcttat ttcaaagtac
caagatattg aaaatagtgc taagagtttc 120 acatatggta tgaccctcta
ttttaagtct cctctaaaga tgaaaagtct tgtgttgaaa 180 ttctcagggt
attttatgag aaataaatga aatttaattt ctctgttttt ccccttttgt 240
aggaagtcac caaagcagta cagcctctct tactgggaag aatcatagct tcctatgacc
300 cggataacaa ggaggaacgc tctatcgcga tttatctagg cataggctta
tgccttctct 360 ttattgtgag gacactgctc ctacacccag ccatttttgg
ccttcatcac attggaatgc 420 agatgagaat agctatgttt agtttgattt
ataagaaggt aatacttcct tgcacaggcc 480 ccatggcaca tatattctgt
atcgtacatg ttttaatgtc ataaattagg tagtgagctg 540 gtacaagtaa
gggataaatg ctgaaattaa tttaatatgc ctattaaata aatggcagga 600
ataattaatg ctcttaatta tccttgataa tttaattgac ttaaactgat aattattgag
660 tatc 664 196 61 DNA Artificial Sequence Diagnostic
Oligonucleotide 196 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
aatttctctg tttttcccct 60 t 61 197 22 DNA Artificial Sequence
Diagnostic Oligonucleotide 197 agctattctc atctgcattc ca 22 198 17
DNA Artificial Sequence Diagnostic Oligonucleotide 198 gacactgctc
ctacacc 17 199 57 DNA Artificial Sequence Diagnostic
Oligonucleotide 199 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tcagcattta tccctta 57 200 17 DNA Artificial Sequence Diagnostic
Oligonucleotide 200 ataacaaatc ccaagtc 17 201 17 DNA Artificial
Sequence Diagnostic
Oligonucleotide 201 tgtaccagct cactacc 17 202 600 DNA Artificial
Sequence Diagnostic Oligonucleotide 202 taattatttc tgcctagatg
ctgggaaata aaacaactag aagcatgcca gtataatatt 60 gactgttgaa
agaaacattt atgaacctga gaagatagta agctagatga atagaatata 120
attttcatta cctttactta ataatgaatg cataataact gaattagtca tattataatt
180 ttacttataa tatatttgta ttttgtttgt tgaaattatc taactttcca
tttttctttt 240 agactttaaa gctgtcaagc cgtgttctag ataaaataag
tattggacaa cttgttagtc 300 tcctttccaa caacctgaac aaatttgatg
aagtatgtac ctattgattt aatcttttag 360 gcactattgt tataaattat
acaactggaa aggcggagtt ttcctgggtc agataatagt 420 aattagtggt
taagtcttgc tcagctctag cttccctatt ctggaaacta agaaaggtca 480
attgtatagc agagcaccat tctggggtct ggtagaacca cccaactcaa aggcacctta
540 gcctgttgtt aataagattt ttcaaaactt aattcttatc agaccttgct
tcttttaaac 600 203 66 DNA Artificial Sequence Diagnostic
Oligonucleotide 203 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ataatatatt tgtattttgt 60 ttgttg 66 204 20 DNA Artificial Sequence
Diagnostic Oligonucleotide 204 aatttgttca ggttgttgga 20 205 18 DNA
Artificial Sequence Diagnostic Oligonucleotide 205 agctgtcaag
ccgtgttc 18 206 59 DNA Artificial Sequence Diagnostic
Oligonucleotide 206 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
atctgaccca ggaaaactc 59 207 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 207 tgctgggaaa taaaac 16 208 16 DNA Artificial
Sequence Diagnostic Oligonucleotide 208 agaatggtgc tctgct 16 209
720 DNA Artificial Sequence Diagnostic Oligonucleotide 209
gacatgatac ttaagatgtc caatcttgat tccactgaat aaaaatatgc ttaaaaatgc
60 actgacttga aatttgtttt ttgggaaaac cgattctatg tgtagaatgt
ttaagcacat 120 tgctatgtgc tccatgtaat gattacctag attttagtgt
gctcagaacc acgaagtgtt 180 tgatcatata agctcctttt acttgctttc
tttcatatat gattgttagt ttctaggggt 240 ggaagataca atgacacctg
tttttgctgt gcttttattt tccagggact tgcattggca 300 catttcgtgt
ggatcgctcc tttgcaagtg gcactcctca tggggctaat ctgggagttg 360
ttacaggcgt ctgccttctg tggacttggt ttcctgatag tccttgccct ttttcaggct
420 gggctaggga gaatgatgat gaagtacagg tagcaaccta ttttcataac
ttgaaagttt 480 taaaaattat gttttcaaaa agcccacttt agtaaaacca
ggactgctct atgcatagaa 540 cagtgatctt cagtgtcatt aaattttttt
tttttttttt tttgagacag agtctagatc 600 tgtcacccag gctggagtgc
agtggcacga tcttggctca ctgcactgca acttctgcct 660 cccaggctca
agcaattctc ctgcctcagc ctccggagta gctgggatta gaggcgcatg 720 210 60
DNA Artificial Sequence Diagnostic Oligonucleotide 210 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ttgttagttt ctaggggtgg 60 211 21
DNA Artificial Sequence Diagnostic Oligonucleotide 211 aaggactatc
aggaaaccaa g 21 212 20 DNA Artificial Sequence Diagnostic
Oligonucleotide 212 gctaatctgg gagttgttac 20 213 62 DNA Artificial
Sequence Diagnostic Oligonucleotide 213 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg agttatgaaa ataggttgct 60 ac 62 214 58 DNA
Artificial Sequence Diagnostic Oligonucleotide 214 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg gggagaatga tgatgaag 58 215 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 215 acactgaaga
tcactgttct a 21 216 18 DNA Artificial Sequence Diagnostic
Oligonucleotide 216 ctccttttac ttgctttc 18 217 17 DNA Artificial
Sequence Diagnostic Oligonucleotide 217 gagcagtcct ggtttta 17 218
480 DNA Artificial Sequence Diagnostic Oligonucleotide 218
atgagtctgt acagcgtctg gcacatagga ggcatttacc aaacagtagt tattattttt
60 gttaccatct atttgataat aaaataatgc ccatctgttg aataaaagaa
atatgactta 120 aaaccttgag cagttcttaa tagataattt gacttgtttt
tactattaga ttgattgatt 180 gattgattga ttgatttaca gagatcagag
agctgggaag atcagtgaaa gacttgtgat 240 tacctcagaa atgattgaaa
atatccaatc tgttaaggca tactgctggg aagaagcaat 300 ggaaaaaatg
attgaaaact taagacagta agttgttcca ataatttcaa tattgttagt 360
aattctgtcc ttaatttttt aaaaatatgt ttatcatggt agacttccac ctcatatttg
420 atgtttgtga caatcaaatg attgcattta agttctgtca atattcatgc
attagttgca 480 219 63 DNA Artificial Sequence Diagnostic
Oligonucleotide 219 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ccttgagcag ttcttaatag 60 ata 63 220 21 DNA Artificial Sequence
Diagnostic Oligonucleotide 220 atgccttaac agattggata t 21 221 21
DNA Artificial Sequence Diagnostic Oligonucleotide 221 gaaaatatcc
aatctgttaa g 21 222 58 DNA Artificial Sequence Diagnostic
Oligonucleotide 222 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tgaggtggaa gtctacca 58 223 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 223 aaaaccttga gcagtt 16 224 17 DNA Artificial
Sequence Diagnostic Oligonucleotide 224 ggtggaagtc taccatg 17 225
752 DNA Artificial Sequence Diagnostic Oligonucleotide 225
tttacaagta ctacaagcaa aacactggta ctttcattgt tatcttttca tataaggtaa
60 ctgaggccca gagagattaa ataacatgcc caaggtcaca caggtcatat
gatgtggagc 120 caggttaaaa atataggcag aaagactcta gagaccatgc
tcagatcttc cattccaaga 180 tccctgatat ttgaaaaata aaataacatc
ctgaatttta ttgttattgt tttttataga 240 acagaactga aactgactcg
gaaggcagcc tatgtgagat acttcaatag ctcagccttc 300 ttcttctcag
ggttctttgt ggtgttttta tctgtgcttc cctatgcact aatcaaagga 360
atcatcctcc ggaaaatatt caccaccatc tcattctgca ttgttctgcg catggcggtc
420 actcggcaat ttccctgggc tgtacaaaca tggtatgact ctcttggagc
aataaacaaa 480 atacaggtaa tgtaccataa tgctgcatta tatactatga
tttaaataat cagtcaatag 540 atcagttcta atgaactttg caaaaatgtg
cgaaaagata gaaaaagaaa tttccttcac 600 taggaagtta taaaagttgc
cagctaatac taggaatgtt caccttaaac ttttcctagc 660 atttctctgg
acagtatgat ggatgagagt ggcatttatg caaattacct taaaatccca 720
ataatactga tgtagctagc agctttgaga aa 752 226 63 DNA Artificial
Sequence Diagnostic Oligonucleotide 226 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg agaccatgct cagatcttcc 60 att 63 227 24 DNA
Artificial Sequence Diagnostic Oligonucleotide 227 gctgccttcc
gagtcagttt cagt 24 228 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 228 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
actgaaactg actcggaagg 60 229 26 DNA Artificial Sequence Diagnostic
Oligonucleotide 229 atggtacatt acctgtattt tgttta 26 230 26 DNA
Artificial Sequence Diagnostic Oligonucleotide 230 ctgtacaaac
atggtatgac tctctt 26 231 65 DNA Artificial Sequence Diagnostic
Oligonucleotide 231 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gtgaaggaaa tttctttttc 60 tatct 65 232 18 DNA Artificial Sequence
Diagnostic Oligonucleotide 232 aatataggca gaaagact 18 233 19 DNA
Artificial Sequence Diagnostic Oligonucleotide 233 gaactgatct
attgactga 19 234 725 DNA Artificial Sequence Diagnostic
Oligonucleotide 234 gcacattagt gggtaattca gggttgcttt gtaaattcat
cactaaggtt agcatgtaat 60 agtacaagga agaatcagtt gtatgttaaa
tctaatgtat aaaaagtttt ataaaatatc 120 atatgtttag agagtatatt
tcaaatatga tgaatcctag tgcttggcaa attaacttta 180 gaacactaat
aaaattattt tattaagaaa taattactat ttcattatta aaattcatat 240
ataagatgta gcacaatgag agtataaagt agatgtaata atgcattaat gctattctga
300 ttctataata tgtttttgct ctcttttata aataggattt cttacaaaag
caagaatata 360 agacattgga atataactta acgactacag aagtagtgat
ggagaatgta acagccttct 420 gggaggaggt cagaattttt aaaaaattgt
ttgctctaaa cacctaactg ttttcttctt 480 tgtgaatatg gatttcatcc
taatggcgaa taaaattaga atgatgatat aactggtaga 540 actggaagga
ggatcactca cttattttct agattaagaa gtagaggaat ggccaggtgc 600
tcatggttgt aatcccagca ctttcgggag accaaggcgg gtggatcacc tgaggtcagg
660 agttcaagac cagcctgcca acatggtaaa acccggtctc tactaaaaat
acaaaaaatt 720 aactg 725 235 61 DNA Artificial Sequence Diagnostic
Oligonucleotide 235 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gcacaatgag agtataaagt 60 a 61 236 21 DNA Artificial Sequence
Diagnostic Oligonucleotide 236 ccatcactac ttctgtagtc g 21 237 27
DNA Artificial Sequence Diagnostic Oligonucleotide 237 ctctctttta
taaataggat ttcttac 27 238 65 DNA Artificial Sequence Diagnostic
Oligonucleotide 238 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ttccagttct accagttata 60 tcatc 65 239 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 239 atgaatccta gtgcttg 17 240 16 DNA
Artificial Sequence Diagnostic Oligonucleotide 240 tccttccagt
tctacc 16 241 876 DNA Artificial Sequence Diagnostic
Oligonucleotide 241 tgtatgtgta tgtatacatg tatgtattca gtctttactg
aaattaaaaa atctttaact 60 tgataatggg caaatatctt agttttagat
catgtcctct agaaaccgta tgctatataa 120 ttatgtacta taaagtaata
atgtatacag tgtaatggat catgggccat gtgcttttca 180 aactaattgt
acataaaaca agcatctatt gaaaatatct gacaaactca tcttttattt 240
ttgatgtgtg tgtgtgtgtg tgtgtgtgtt tttttaacag ggatttgggg aattatttga
300 gaaagcaaaa caaaacaata acaatagaaa aacttctaat ggtgatgaca
gcctcttctt 360 cagtaatttc tcacttcttg gtactcctgt cctgaaagat
attaatttca agatagaaag 420 aggacagttg ttggcggttg ctggatccac
tggagcaggc aaggtagttc ttttgttctt 480 cactattaag aacttaattt
ggtgtccatg tctctttttt tttctagttt gtagtgctgg 540 aaggtatttt
tggagaaatt cttacatgag cattaggaga atgtatgggt gtagtgtctt 600
gtataataga aattgttcca ctgataattt actctagttt tttatttcct catattattt
660 tcagtggctt tttcttccac atctttatat tttgcaccac attcaacact
gtatcttgca 720 catggcgagc attcaataac tttattgaat aaacaaatca
tccattttat ccattcttaa 780 ccagaacaga cattttttca gagctggtcc
aggaaaatca tgacttacat tttgccttag 840 taaccacata aacaaaaagt
ctccattttt gttgac 876 242 65 DNA Artificial Sequence Diagnostic
Oligonucleotide 242 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
acaatagaaa aacttctaat 60 ggtga 65 243 22 DNA Artificial Sequence
Diagnostic Oligonucleotide 243 aaaaaagaga catggacacc aa 22 244 16
DNA Artificial Sequence Diagnostic Oligonucleotide 244 agaaaccgta
tgctat 16 245 17 DNA Artificial Sequence Diagnostic Oligonucleotide
245 cccatacatt cttccta 17 246 17 DNA Artificial Sequence Diagnostic
Oligonucleotide 246 taaagatgtg gaagaaa 17 247 831 DNA Artificial
Sequence Diagnostic Oligonucleotide 247 cactgtagct gtactacctt
ccatctcctc aacctattcc aactatctga atcatgtgcc 60 cttctctgtg
aacctctatc ataatacttg tcacactgta ttgtaattgt ctcttttact 120
ttcccttgta tcttttgtgc atagcagagt acctgaaaca ggaagtattt taaatatttt
180 gaatcaaatg agttaataga atctttacaa ataagaatat acacttctgc
ttaggatgat 240 aattggaggc aagtgaatcc tgagcgtgat ttgataatga
cctaataatg atgggtttta 300 tttccagact tcacttctaa tgatgattat
gggagaactg gagccttcag agggtaaaat 360 taagcacagt ggaagaattt
cattctgttc tcagttttcc tggattatgc ctggcaccat 420 taaagaaaat
atcatctttg gtgtttccta tgatgaatat agatacagaa gcgtcatcaa 480
agcatgccaa ctagaagagg taagaaacta tgtgaaaact ttttgattat gcatatgaac
540 ccttcacact acccaaatta tatatttggc tccatattca atcggttagt
ctacatatat 600 ttatgtttcc tctatgggta agctactgtg aatggatcaa
ttaataaaac acatgaccta 660 tgctttaaga agcttgcaaa cacatgaaat
aaatgcaatt tattttttaa ataatgggtt 720 catttgatca caataaatgc
attttatgaa atggtgagaa ttttgttcac tcattagtga 780 gacaaacgtc
tcaatggtta tttatatggc atgcatatag tgatatgtgg t 831 248 18 DNA
Artificial Sequence Diagnostic Oligonucleotide 248 cctgagcgtg
atttgata 18 249 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 249 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
atgtagacta accgattgaa 60 250 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 250 gggagaactg gagcct 16 251 58 DNA Artificial
Sequence Diagnostic Oligonucleotide 251 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg aaccgattga atatggag 58 252 17 DNA Artificial
Sequence Diagnostic Oligonucleotide 252 ccttgtatct tttgtgc 17 253
16 DNA Artificial Sequence Diagnostic Oligonucleotide 253
ccgattgaat atggag 16 254 614 DNA Artificial Sequence Diagnostic
Oligonucleotide 254 atatacccat aaatatacac atattttaat ttttggtatt
ttataattat tatttaatga 60 tcattcatga cattttaaaa attacaggaa
aaatttacat ctaaaatttc agcaatgttg 120 tttttgacca actaaataaa
ttgcatttga aataatggag atgcaatgtt caaaatttca 180 actgtggtta
aagcaatagt gtgatatatg attacattag aaggaagatg tgcctttcaa 240
attcagattg agcatactaa aagtgactct ctaattttct atttttggta ataggacatc
300 tccaagtttg cagagaaaga caatatagtt cttggagaag gtggaatcac
actgagtgga 360 ggtcaacgag caagaatttc tttagcaagg tgaataacta
attattggtc tagcaagcat 420 ttgctgtaaa tgtcattcat gtaaaaaaat
tacagacatt tctctattgc tttatattct 480 gtttctggaa ttgaaaaaat
cctggggttt tatggctagt gggttaagaa tcacatttaa 540 gaactataaa
taatggtata gtatccagat ttggtagaga ttatggttac tcagaatctg 600
tgcccgtatc ttgg 614 255 61 DNA Artificial Sequence Diagnostic
Oligonucleotide 255 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gatatatgat tacattagaa 60 g 61 256 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 256 accttctcca agaacta 17 257 18 DNA
Artificial Sequence Diagnostic Oligonucleotide 257 ataggacatc
tccaagtt 18 258 59 DNA Artificial Sequence Diagnostic
Oligonucleotide 258 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gcaatagaga aatgtctgt 59 259 20 DNA Artificial Sequence Diagnostic
Oligonucleotide 259 cagattgagc atactaaaag 20 260 16 DNA Artificial
Sequence Diagnostic Oligonucleotide 260 aagatacggg cacaga 16 261
676 DNA Artificial Sequence Diagnostic Oligonucleotide 261
cttacagtta gcaaaatcac ttcagcagtt cttggaatgt tgtgaaaagt gataaaaatc
60 ttctgcaact tattccttta ttcctcattt aaaataatct accatagtaa
aaacatgtat 120 aaaagtgcta cttctgcacc acttttgaga atagtgttat
ttcagtgaat cgatgtggtg 180 accatattgt aatgcatgta gtgaactgtt
taaggcaaat catctacact agatgaccag 240 gaaatagaga ggaaatgtaa
tttaatttcc attttctttt tagagcagta tacaaagatg 300 ctgatttgta
tttattagac tctccttttg gatacctaga tgttttaaca gaaaaagaaa 360
tatttgaaag gtatgttctt tgaatacctt acttataatg ctcatgctaa aataaaagaa
420 agacagactg tcccatcata gattgcattt tacctcttga gaaatatgtt
caccattgtt 480 ggtatggcag aatgtagcat ggtattaact caaatctgat
ctgccctact gggccaggat 540 tcaagattac ttccattaaa accttttctc
accgcctcat gctaaaccag tttctctcat 600 tgctatactg ttatagcaat
tgctatctat gtagtttttg cagtatcatt gccttgtgat 660 atatattact ttaatt
676 262 63 DNA Artificial Sequence Diagnostic Oligonucleotide 262
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg gtgaactgtt taaggcaaat
60 cat 63 263 21 DNA Artificial Sequence Diagnostic Oligonucleotide
263 tgatgggaca gtctgtcttt c 21 264 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 264 tcacttcagc agttctt 17 265 17 DNA
Artificial Sequence Diagnostic Oligonucleotide 265 caatctatga
tgggaca 17 266 1200 DNA Artificial Sequence Diagnostic
Oligonucleotide 266 gaattcacaa ggtaccaatt taattactac agagtactta
tagaatcatt taaaatataa 60 taaaattgta tgatagagat tatatgcaat
aaaacattaa caaaatgcta aaatacgaga 120 catattgcaa taaagtattt
ataaaattga tatttatatg tttttatatc ttaaagctgt 180 gtctgtaaac
tgatggctaa caaaactagg attttggtca cttctaaaat ggaacattta 240
aagaaagctg acaaaatatt aattttgcat gaaggtagca gctattttta tgggacattt
300 tcagaactcc aaaatctaca gccagacttt agctcaaaac tcatgggatg
tgattctttc 360 gaccaattta gtgcagaaag aagaaattca atcctaactg
agaccttaca ccgtttctca 420 ttagaaggag atgctcctgt ctcctggaca
gaaacaaaaa aacaatcttt taaacagact 480 ggagagtttg gggaaaaaag
gaagaattct attctcaatc caatcaactc tatacgaaaa 540 ttttccattg
tgcaaaagac tcccttacaa atgaatggca tcgaagagga ttctgatgag 600
cctttagaga gaaggctgtc cttagtacca gattctgagc agggagaggc gatactgcct
660 cgcatcagcg tgatcagcac tggccccacg cttcaggcac
gaaggaggca gtctgtcctg 720 aacctgatga cacactcagt taaccaaggt
cagaacattc accgaaagac aacagcatcc 780 acacgaaaag tgtcactggc
ccctcaggca aacttgactg aactggatat atattcaaga 840 aggttatctc
aagaaactgg cttggaaata agtgaagaaa ttaacgaaga agacttaaag 900
gtaggtatac atcgcttggg ggtatttcac cccacagaat gcaattgagt agaatgcaat
960 atgtagcatg taacaaaatt tactaaaatc ataggattag gataaggtgt
atcttaaaac 1020 tcagaaagta tgaagttcat taattataca agcaacgtta
aaatgtaaaa taacaaatga 1080 tttctttttg caatggacat atctcttccc
ataaaatggg aaaggattta gtttttggtc 1140 ctctactaag ccagtgataa
ctgtgactat agttagaaag catttgcttt attaccatct 1200 267 25 DNA
Artificial Sequence Diagnostic Oligonucleotide 267 aatacgagac
atattgcaat aaagt 25 268 62 DNA Artificial Sequence Diagnostic
Oligonucleotide 268 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ctggctgtag attttggagt 60 tc 62 269 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 269 aggtagcagc tattttt 17 270 57 DNA
Artificial Sequence Diagnostic Oligonucleotide 270 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ggacagcctt ctctcta 57 271 61 DNA
Artificial Sequence Diagnostic Oligonucleotide 271 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg atgggacatt ttcagaactc 60 c 61 272
19 DNA Artificial Sequence Diagnostic Oligonucleotide 272
cctcttcgat gccattcat 19 273 63 DNA Artificial Sequence Diagnostic
Oligonucleotide 273 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
caatccaatc aactctatac 60 gaa 63 274 18 DNA Artificial Sequence
Diagnostic Oligonucleotide 274 ctgatcacgc tgatgcga 18 275 60 DNA
Artificial Sequence Diagnostic Oligonucleotide 275 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg tgatgagcct ttagagagaa 60 276 19
DNA Artificial Sequence Diagnostic Oligonucleotide 276 ccagttcagt
caagtttgc 19 277 54 DNA Artificial Sequence Diagnostic
Oligonucleotide 277 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
cagcgtgatc agca 54 278 18 DNA Artificial Sequence Diagnostic
Oligonucleotide 278 tttgttacat gctacata 18 279 734 DNA Artificial
Sequence Diagnostic Oligonucleotide 279 ggaaacttca tttagatggt
atcattcatt tgataaaagg tatgccactg ttaagccttt 60 aatggtaaaa
ttgtccaata ataatacagt tatataatca gtgatacatt tttagaattt 120
tgaaaaatta cgatgtttct catttttaat aaagctgtgt tgctccagta gacattattc
180 tggctataga atgacatcat acatggcatt tataatgatt tatatttgtt
aaaatacact 240 tagattcaag taatactatt cttttatttt catatattaa
aaataaaacc acaatggtgg 300 catgaaactg tactgtctta ttgtaatagc
cataattctt ttattcagga gtgctttttt 360 gatgatatgg agagcatacc
agcagtgact acatggaaca cataccttcg atatattact 420 gtccacaaga
gcttaatttt tgtgctaatt tggtgcttag taatttttct ggcagaggta 480
agaatgttct attgtaaagt attactggat ttaaagttaa attaagatag tttggggatg
540 tatacatata tatgcacaca cataaatatg tatatataca catgtataca
tgtataagta 600 tgcatatata cacacatata tcactatatg tatatatgta
tatattacat atatttgtga 660 ttttacagta tataatggta tagattcata
tagttcttag cttctgaaaa atcaacaagt 720 agaaccacta ctga 734 280 62 DNA
Artificial Sequence Diagnostic Oligonucleotide 280 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ttcatatatt aaaaataaaa 60 cc 62 281
21 DNA Artificial Sequence Diagnostic Oligonucleotide 281
taatatatcg aaggtatgtg t 21 282 22 DNA Artificial Sequence
Diagnostic Oligonucleotide 282 gagcatacca gcagtgacta ca 22 283 69
DNA Artificial Sequence Diagnostic Oligonucleotide 283 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg gtaatacttt acaatagaac 60 attcttacc
69 284 20 DNA Artificial Sequence Diagnostic Oligonucleotide 284
accagcagtg actacatgga 20 285 28 DNA Artificial Sequence Diagnostic
Oligonucleotide 285 atatttatgt gtgtgcatat atatgtat 28 286 17 DNA
Artificial Sequence Diagnostic Oligonucleotide 286 tgttgctcca
gtagaca 17 287 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 287 catccccaaa ctatct 16 288 621 DNA Artificial
Sequence Diagnostic Oligonucleotide 288 gaattccatt aacttaatgt
ggtctcatca caaataatag tacttagaac acctagtaca 60 gctgctggac
ccaggaacac aaagcaaagg aagatgaaat tgtgtgtacc ttgatattgg 120
tacacacatc aaatggtgtg atgtgaattt agatgtgggc atgggaggaa taggtgaaga
180 tgttagaaaa aaaatcaact gtgtcttgtt ccattccagg tggctgcttc
tttggttgtg 240 ctgtggctcc ttggaaagtg agtattccat gtcctattgt
gtagattgtg ttttatttct 300 gttgattaaa tattgtaatc cactatgttt
gtatgtattg taatccactt tgtttcattt 360 ctcccaagca ttatggtagt
ggaaagataa ggttttttgt ttaaatgatg accattagtt 420 gggtgaggtg
acacattcct gtagtcctag ctcctccaca ggctgacgca ggaggatcac 480
ttgagcccag gagttcaggg ctgtagtgtt gtatcattgt gagtagccac caccgcactc
540 cagcctggac aatatagtga gatcctatat ctaaaataaa ataaaataaa
atgaataaat 600 tgtgagcatg tgcagctcct g 621 289 57 DNA Artificial
Sequence Diagnostic Oligonucleotide 289 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg gtgtaccttg atattgg 57 290 16 DNA Artificial
Sequence Diagnostic Oligonucleotide 290 ctcactttcc aaggag 16 291 15
DNA Artificial Sequence Diagnostic Oligonucleotide 291 gctgtggctc
cttgg 15 292 58 DNA Artificial Sequence Diagnostic Oligonucleotide
292 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg actacagccc tgaactcc
58 293 16 DNA Artificial Sequence Diagnostic Oligonucleotide 293
ggaacacaaa gcaaag 16 294 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 294 tgggagaaat gaaaca 16 295 727 DNA Artificial
Sequence Diagnostic Oligonucleotide 295 tcctatatct aaataaataa
ataaatgaat aaattgtgag catgtgcagc tcctgcagtt 60 tctaaagaat
atagttctgt tcagtttctg tgaaacacaa taaaaatatt tgaaataaca 120
ttacatattt agggttttct tcaaattttt taatttaata aagaacaact caatctctat
180 caatagtgag aaaacatatc tattttcttg caataatagt atgattttga
ggttaagggt 240 gcatgctctt ctaatgcaaa atattgtatt tatttagact
caagtttagt tccatttaca 300 tgtattggaa attcagtaag taactttggc
tgccaaataa cgatttccta tttgctttac 360 agcactcctc ttcaagacaa
agggaatagt actcatagta gaaataacag ctatgcagtg 420 attatcacca
gcaccagttc gtattatgtg ttttacattt acgtgggagt agccgacact 480
ttgcttgcta tgggattctt cagaggtcta ccactggtgc atactctaat cacagtgtcg
540 aaaattttac accacaaaat gttacattct gttcttcaag cacctatgtc
aaccctcaac 600 acgttgaaag caggtacttt actaggtcta agaaatgaaa
ctgctgatcc accatcaata 660 gggcctgtgg ttttgttggt tttctaatgg
cagtgctggc ttttgcacag aggcatgtgc 720 ctttgtt 727 296 66 DNA
Artificial Sequence Diagnostic Oligonucleotide 296 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg catgtattgg aaattcagta 60 agtaac 66
297 24 DNA Artificial Sequence Diagnostic Oligonucleotide 297
ttcgacactg tgattagagt atgc 24 298 16 DNA Artificial Sequence
Diagnostic Oligonucleotide 298 gtgggagtag ccgaca 16 299 57 DNA
Artificial Sequence Diagnostic Oligonucleotide 299 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg caggccctat tgatggt 57 300 16 DNA
Artificial Sequence Diagnostic Oligonucleotide 300 cgtgggagta
gccgac 16 301 58 DNA Artificial Sequence Diagnostic Oligonucleotide
301 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg cattagaaaa ccaacaaa
58 302 19 DNA Artificial Sequence Diagnostic Oligonucleotide 302
agactcaagt ttagttcca 19 303 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 303 ccaacaaaac cacagg 16 304 701 DNA Artificial
Sequence Diagnostic Oligonucleotide 304 gtaagattgt aagcaggatg
agtacccacc tattcctgac ataatttata gtaaaagcta 60 tttcagagaa
attggtcgtt acttgaatct tacaagaatc tgaaactttt aaaaaggttt 120
aaaagtaaaa gacaataact tgaacacata attatttaga atgtttggaa agaaacaaaa
180 atttctaagt ctatctgatt ctatttgcta attcttattt gggttctgaa
tgcgtctact 240 gtgatccaaa cttagtattg aatatattga tatatcttta
aaaaattagt gttttttgag 300 gaatttgtca tcttgtatat tataggtggg
attcttaata gattctccaa agatatagca 360 attttggatg accttctgcc
tcttaccata tttgacttca tccaggtatg taaaaataag 420 taccgttaag
tatgtctgta ttattaaaaa aacaataaca aaagcaaatg tgattttgtt 480
ttcatttttt atttgattga gggttgaagt cctgtctatt gcattaattt tgtaattatc
540 caaagccttc aaaatagaca taagtttagt aaattcaata ataagtcaga
actgcttacc 600 tggcccaaac ctgaggcaat cccacattta gatgtaatag
ctgtctactt gggagtgatt 660 tgagaggcac aaaggaccat ctttcccaaa
atcactggca c 701 305 56 DNA Artificial Sequence Diagnostic
Oligonucleotide 305 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ctgaatgcgt ctactg 56 306 17 DNA Artificial Sequence Diagnostic
Oligonucleotide 306 catccaaaat tgctata 17 307 23 DNA Artificial
Sequence Diagnostic Oligonucleotide 307 ttgaggaatt tgtcatcttg tat
23 308 63 DNA Artificial Sequence Diagnostic Oligonucleotide 308
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg caaaatcaca tttgcttttg
60 tta 63 309 17 DNA Artificial Sequence Diagnostic Oligonucleotide
309 atgcgtctac tgtgatc 17 310 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 310 cttcaaccct caatca 16 311 637 DNA Artificial
Sequence Diagnostic Oligonucleotide 311 agtgcaccag catggcacat
gtatacatat gtaactaacc tcgacaatgt gcacatgtac 60 cctaaaactt
aaagtataat aaaaaaaata aaaaaaagtt tgaggtgttt aaagtatgca 120
aaaaaaaaaa aagaaataaa tcactgacac actttgtcca ctttgcaatg tgaaaatgtt
180 tactcaccaa catgttttct ttgatcttac agttgttatt aattgtgatt
ggagctatag 240 cagttgtcgc agttttacaa ccctacatct ttgttgcaac
agtgccagtg atagtggctt 300 ttattatgtt gagagcatat ttcctccaaa
cctcacagca actcaaacaa ctggaatctg 360 aaggtatgac agtgaatgtg
cgatactcat cttgtaaaaa agctataaga gctatttgag 420 attctttatt
gttaatctac ttaaaaaaaa ttctgctttt aaacttttac atcatataac 480
aataattttt ttctacatgc atgtgtatat aaaaggaaac tatattacaa agtacacatg
540 gatttttttt cttaattaat gaccatgtga cttcattttg gttttaaaat
aggtatatag 600 aatcttacca cagttggtgt acaggacatt catttat 637 312 58
DNA Artificial Sequence Diagnostic Oligonucleotide 312 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg aaagaaataa atcactga 58 313 16 DNA
Artificial Sequence Diagnostic Oligonucleotide 313 gtaaaactgc
gacaac 16 314 26 DNA Artificial Sequence Diagnostic Oligonucleotide
314 ccaacatgtt ttctttgatc ttacag 26 315 67 DNA Artificial Sequence
Diagnostic Oligonucleotide 315 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg agaatctcaa atagctctta 60 tagcttt 67 316 19 DNA
Artificial Sequence Diagnostic Oligonucleotide 316 aaataaatca
ctgacacac 19 317 17 DNA Artificial Sequence Diagnostic
Oligonucleotide 317 aatgaagtca catggtc 17 318 600 DNA Artificial
Sequence Diagnostic Oligonucleotide 318 ttcaaagaat ggcaccagtg
tgaaaaaaag ctttttaacc aatgacattt gtgatatgat 60 tattctaatt
tagtcttttt caggtacaag atattatgaa aattacattt tgtgtttatg 120
ttatttgcaa tgttttctat ggaaatattt cacaggcagg agtccaattt tcactcatct
180 tgttacaagc ttaaaaggac tatggacact tcgtgccttc ggacggcagc
cttactttga 240 aactctgttc cacaaagctc tgaatttaca tactgccaac
tggttcttgt acctgtcaac 300 actgcgctgg ttccaaatga gaatagaaat
gatttttgtc atcttcttca ttgctgttac 360 cttcatttcc attttaacaa
caggtactat gaactcatta actttagcta agcatttaag 420 taaaaaattt
tcaatgaata aaatgctgca ttctataggt tatcaatttt tgatatcttt 480
agagtttagt aattaacaaa tttgttggtt tattattgaa caagtgattt ctttgaaatt
540 tccattgttt tattgttaaa caaataattt ccttgaaatc ggtatatata
tatatatagt 600 319 62 DNA Artificial Sequence Diagnostic
Oligonucleotide 319 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ttaaccaatg acatttgtga 60 ta 62 320 21 DNA Artificial Sequence
Diagnostic Oligonucleotide 320 gtgtccatag tccttttaag c 21 321 56
DNA Artificial Sequence Diagnostic Oligonucleotide 321 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg aatatttcac aggcag 56 322 16 DNA
Artificial Sequence Diagnostic Oligonucleotide 322 tgaaggtaac
agcaat 16 323 17 DNA Artificial Sequence Diagnostic Oligonucleotide
323 acttcgtgcc ttcggac 17 324 62 DNA Artificial Sequence Diagnostic
Oligonucleotide 324 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
cagcaatgaa gaagatgaca 60 aa 62 325 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 325 ctggttccaa atgagaa 17 326 59 DNA
Artificial Sequence Diagnostic Oligonucleotide 326 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg taacctatag aatgcagca 59 327 739
DNA Artificial Sequence Diagnostic Oligonucleotide 327 ttattactta
tagaataata gtagaagaga caaatatggt acctacccat taccaacaac 60
acctccaata ccagtaacat tttttaaaaa gggcaacact ttcctaatat tcaatcgctc
120 tttgatttaa aatcctggtt gaatacttac tatatgcaga gcattattct
attagtagat 180 gctgtgatga actgagattt aaaaattgtt aaaattagca
taaaattgaa atgtaaattt 240 aatgtgatat gtgccctagg agaagtgtga
ataaagtcgt tcacagaaga gagaaataac 300 atgaggttca tttacgtctt
ttgtgcatct ataggagaag gagaaggaag agttggtatt 360 atcctgactt
tagccatgaa tatcatgagt acattgcagt gggctgtaaa ctccagcata 420
gatgtggata gcttggtaag tcttatcatc tttttaactt ttatgaaaaa aattcagaca
480 agtaacaaag tatgagtaat agcatgagga agaactatat accgtatatt
gagcttaaga 540 aataaaacat tacagataaa ttgagggtca ctgtgtatct
gtcattaaat ccttatctct 600 tctttccttc tcatagatag ccactatgaa
gatctaatac tgcagtgagc attctttcac 660 ctgtttcctt attcaggatt
ttctaggaga aatacctagg ggttgtattg ctgggtcata 720 ggattcaccc
atgcttaac 739 328 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 328 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ttaatgtgat atgtgcccta 60 329 21 DNA Artificial Sequence Diagnostic
Oligonucleotide 329 agatgataag acttaccaag c 21 330 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 330 gagaaggaga
aggaagagtt g 21 331 63 DNA Artificial Sequence Diagnostic
Oligonucleotide 331 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
cttcctcatg ctattactca 60 tac 63 332 18 DNA Artificial Sequence
Diagnostic Oligonucleotide 332 cctggttgaa tacttact 18 333 20 DNA
Artificial Sequence Diagnostic Oligonucleotide 333 ctcatacttt
gttacttgtc 20 334 629 DNA Artificial Sequence Diagnostic
Oligonucleotide 334 ttctcttcag ttaaactttt aattatatcc aattatttcc
tgttagttca ttgaaaagcc 60 cgacaaataa ccaagtgaca aatagcaagt
gttgcatttt acaagttatt ttttaggaag 120 catcaaacta attgtgaaat
tgtctgccat tcttaaaaac aaaaatgttg ttatttttat 180 ttcagatgcg
atctgtgagc cgagtcttta agttcattga catgccaaca gaaggtaaac 240
ctaccaagtc aaccaaacca tacaagaatg gccaactctc gaaagttatg attattgaga
300 attcacacgt gaagaaagat gacatctggc cctcaggggg ccaaatgact
gtcaaagatc 360 tcacagcaaa atacacagaa ggtggaaatg ccatattaga
gaacatttcc ttctcaataa 420 gtcctggcca gagggtgaga tttgaacact
gcttgctttg ttagactgtg ttcagtaagt 480 gaatcccagt agcctgaagc
aatgtgttag cagaatctat ttgtaacatt attattgtac 540 agtagaatca
atattaaaca cacatgtttt attatatgga gtcattattt ttaatatgaa 600
atttaatttg cagagtctga actatatat 629 335 61 DNA Artificial Sequence
Diagnostic Oligonucleotide 335 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg aagttatttt ttaggaagca 60 t 61 336 19 DNA Artificial
Sequence Diagnostic Oligonucleotide 336 gaacttaaag actcggctc 19 337
61 DNA Artificial Sequence Diagnostic Oligonucleotide 337
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg gaaattgtct gccattctta
60 a 61 338 21 DNA Artificial Sequence Diagnostic Oligonucleotide
338 gagttggcca ttctttgtat g
21 339 56 DNA Artificial Sequence Diagnostic Oligonucleotide 339
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg tgtgagccga gtcttt 56
340 16 DNA Artificial Sequence Diagnostic Oligonucleotide 340
atggcatttc cacctt 16 341 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 341 cgtgaagaaa gatgac 16 342 59 DNA Artificial
Sequence Diagnostic Oligonucleotide 342 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg taatgttaca aatagattc 59 343 18 DNA Artificial
Sequence Diagnostic Oligonucleotide 343 gacaaataac caagtgac 18 344
16 DNA Artificial Sequence Diagnostic Oligonucleotide 344
aacacattgc ttcagg 16 345 720 DNA Artificial Sequence Diagnostic
Oligonucleotide 345 acttaactgc tttctccatt tgtagtctct tgaaaataca
gaaatttcag aaataattta 60 taagaatatc aaggattcaa atcatatcag
cacaaacacc taaatacttg tttgctttgt 120 taaacacata tcccattttc
tatcttgata aacattggtg taaagtagtt gaatcattca 180 gtgggtataa
gcagcatatt ctcaatacta tgtttcatta ataattaata gagatatatg 240
aacacataaa agattcaatt ataatcacct tgtggatcta aatttcagtt gacttgtcat
300 cttgatttct ggagaccaca aggtaatgaa aaataattac aagagtcttc
catctgttgc 360 agtattaaaa tggcgagtaa gacaccctga aaggaaatgt
tctattcatg gtacaatgca 420 attacagcta gcaccaaatt caacactgtt
taactttcaa catattattt tgatttatct 480 tgatccaaca ttctcaggga
ggaggtgcat tgaagttatt agaaaacact gacttagatt 540 tagggtatgt
cttaaaagct tatttgcggg aagtactcta gccttattca acagatcact 600
gagaagcctg gaaaaacaaa tcccggaaac taattattat gtgccagtta tataaacaag
660 aagactttgt tgggtacaaa ccagtgattc cttgcctttg aaaaatgtgt
cagatatcat 720 346 56 DNA Artificial Sequence Diagnostic
Oligonucleotide 346 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
cttgatttct ggagac 56 347 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 347 ctagctgtaa ttgcat 16 348 16 DNA Artificial
Sequence Diagnostic Oligonucleotide 348 agtgggtata agcagc 16 349 19
DNA Artificial Sequence Diagnostic Oligonucleotide 349 gttgaataag
gctagagta 19 350 600 DNA Artificial Sequence Diagnostic
Oligonucleotide 350 aaaggtcagt gataaaggaa gtctgcatca ggggtccaat
tccttatggc cagtttctct 60 attctgttcc aaggttgttt gtctccatat
atcaacattg gtcaggattg aaagtgtgca 120 acaaggtttg aatgaataag
tgaaaatctt ccactggtga caggataaaa tattccaatg 180 gtttttattg
aagtacaata ctgaattatg tttatggcat ggtacctata tgtcacagaa 240
gtgatcccat cacttttacc ttataggtgg gcctcttggg aagaactgga tcagggaaga
300 gtactttgtt atcagctttt ttgagactac tgaacactga aggagaaatc
cagatcgatg 360 gtgtgtcttg ggattcaata actttgcaac agtggaggaa
agcctttgga gtgataccac 420 aggtgagcaa aaggacttag ccagaaaaaa
ggcaactaaa ttatattttt tactgctatt 480 tgatacttgt actcaagaaa
ttcatattac tctgcaaaat atatttgtta tgcattgctg 540 tctttttttt
ctccagtgca gttttctcat aggcagaaaa gatgtctcta aaagtttggg 600 351 61
DNA Artificial Sequence Diagnostic Oligonucleotide 351 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg gaattatgtt tatggcatgg 60 t 61 352
25 DNA Artificial Sequence Diagnostic Oligonucleotide 352
gagtacaagt atcaaatagc agtaa 25 353 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 353 aaatcttcca ctggtga 17 354 18 DNA
Artificial Sequence Diagnostic Oligonucleotide 354 gacatctttt
ctgcctat 18 355 59 DNA Artificial Sequence Diagnostic
Oligonucleotide 355 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ctgaattatg tttatggca 59 356 17 DNA Artificial Sequence Diagnostic
Oligonucleotide 356 cttttttctg gctaagt 17 357 716 DNA Artificial
Sequence Diagnostic Oligonucleotide 357 tttttaatat tctacaatta
acaattatct caatttcttt attctaaaga cattggatta 60 gaaaaatgtt
cacaagggac tccaaatatt gctgtagtat ttgtttctta aaagaatgat 120
acaaagcaga catgataaaa tattaaaatt tgagagaact tgatggtaag tacatgggtg
180 tttcttattt taaaataatt tttctacttg aaatatttta caatacaata
agggaaaaat 240 aaaaagttat ttaagttatt catactttct tcttcttttc
ttttttgcta tagaaagtat 300 ttattttttc tggaacattt agaaaaaact
tggatcccta tgaacagtgg agtgatcaag 360 aaatatggaa agttgcagat
gaggtaaggc tgctaactga aatgattttg aaaggggtaa 420 ctcataccaa
cacaaatggc tgatatagct gacatcattc tacacacttt gtgtgcatgt 480
atgtgtgtgc acaactttaa aatggagtac cctaacatac ctggagcaac aggtactttt
540 gactggacct acccctaact gaaatgattt tgaaagaggt aactcatacc
aacacaaatg 600 gttgatatgg ctaagatcat tctacacact ttgtgtgcat
gtatttctgt gcacaacttc 660 aaaatggagt accctaaaat acctggcgcg
acaagtactt ttgactgagc ctactt 716 358 20 DNA Artificial Sequence
Diagnostic Oligonucleotide 358 atggtaagta catgggtgtt 20 359 22 DNA
Artificial Sequence Diagnostic Oligonucleotide 359 ccactgttca
tagggatcca ag 22 360 56 DNA Artificial Sequence Diagnostic
Oligonucleotide 360 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tttctggaac atttag 56 361 18 DNA Artificial Sequence Diagnostic
Oligonucleotide 361 gaatgatgtc agctatat 18 362 16 DNA Artificial
Sequence Diagnostic Oligonucleotide 362 tgttcacaag ggactc 16 363 16
DNA Artificial Sequence Diagnostic Oligonucleotide 363 cagttagggg
taggtc 16 364 60 DNA Artificial Sequence Diagnostic Oligonucleotide
364 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg agttattcat
actttcttct 60 365 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 365 agccttacct catctg 16 366 910 DNA Artificial
Sequence Diagnostic Oligonucleotide 366 cacagttgac tattttatgc
tatcttttgt cctcagtcat gacagagtag aagatgggag 60 gtagcaccaa
ggatgatgtc atacctccat cctttatgct acattctatc ttctgtctac 120
ataagatgtc atactagagg gcatatctgc aatgtataca tattatcttt tccagcatgc
180 attcagttgt gttggaataa tttatgtaca cctttataaa cgctgagcct
cacaagagcc 240 atgtgccacg tattgtttct tactactttt ggatacctgg
cacgtaatag acactcattg 300 aaagtttcct aatgaatgaa gtacaaagat
aaaacaagtt atagactgat tcttttgagc 360 tgtcaaggtt gtaaatagac
ttttgctcaa tcaattcaaa tggtggcagg tagtgggggt 420 agagggattg
gtatgaaaaa cataagcttt cagaactcct gtgtttattt ttagaatgtc 480
aactgcttga gtgtttttaa ctctgtggta tctgaactat cttctctaac tgcaggttgg
540 gctcagatct gtgatagaac agtttcctgg gaagcttgac tttgtccttg
tggatggggg 600 ctgtgtccta agccatggcc acaagcagtt gatgtgcttg
gctagatctg ttctcagtaa 660 ggcgaagatc ttgctgcttg atgaacccag
tgctcatttg gatccagtgt gagtttcaga 720 tgttctgtta cttaatagca
cagtgggaac agaatcatta tgcctgcttc atggtgacac 780 atatttctat
taggctgtca tgtctgcgtg tgggggtctc ccaagatatg aaataattgc 840
ccagtggaaa tgagcataaa tgcatatttc cttgctaaga gttcttgtgt tttcttccga
900 agatagtttt 910 367 58 DNA Artificial Sequence Diagnostic
Oligonucleotide 367 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tgagctgtca aggttgta 58 368 19 DNA Artificial Sequence Diagnostic
Oligonucleotide 368 caggaaactg ttctatcac 19 369 64 DNA Artificial
Sequence Diagnostic Oligonucleotide 369 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg gaatgtcaac tgcttgagtg 60 tttt 64 370 27 DNA
Artificial Sequence Diagnostic Oligonucleotide 370 aagtaacaga
acatctgaaa ctcacac 27 371 17 DNA Artificial Sequence Diagnostic
Oligonucleotide 371 cttgctgctt gatgaac 17 372 20 DNA Artificial
Sequence Diagnostic Oligonucleotide 372 gcaattattt catatcttgg 20
373 17 DNA Artificial Sequence Diagnostic Oligonucleotide 373
agggattggt atgaaaa 17 374 18 DNA Artificial Sequence Diagnostic
Oligonucleotide 374 ggaagaaaac acaagaac 18 375 840 DNA Artificial
Sequence Diagnostic Oligonucleotide 375 gcatgtttat agccccaaat
aaaagaagta ctggtgattc tacataatga aaatgtactc 60 atttattaaa
gtttctttga aatatttgtc ctgtttattt atggatactt agagtctacc 120
ccatggttga aaagctgatt gtgcgtaacg ctatatcaac attatgtgaa aagaacttaa
180 agaaataagt aatttaaaga gataatagaa caatagacat attatcaagg
taaatacaga 240 tcattactgt tctgtgatat tatgtgtggt attttctttc
ttttctagaa cataccaaat 300 aattagaaga actctaaaac aagcatttgc
tgattgcaca gtaattctct gtgaacacag 360 gatagaagca atgctggaat
gccaacaatt tttggtgagt ctttataact ttacttaaga 420 tctcattgcc
cttgtaattc ttgataacaa tctcacatgt gatagttcct gcaaattgca 480
acaatgtaca agttcttttc aaaaatatgt atcatacagc catccagctt tactcaaaat
540 agctgcacaa gtttttcact ttgatctgag ccatgtggtg aggttgaaat
atagtaaatc 600 taaaatggca gcatattact aagttatgtt tataaatagg
atatatatac ttttgagccc 660 tttatttggg accaagtcat acaaaatact
ctactgttta agattttaaa aaaggtccct 720 gtgattcttt caataactaa
atgtcccatg gatgtggtct ggacaggcct agttgtctta 780 cagtctgatt
tatggtatta atgacaaagt tgagaggcac atttcatttt tctagccatg 840 376 58
DNA Artificial Sequence Diagnostic Oligonucleotide 376 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg tatcaaggta aatacaga 58 377 17 DNA
Artificial Sequence Diagnostic Oligonucleotide 377 gcttctatcc
tgtgttc 17 378 21 DNA Artificial Sequence Diagnostic
Oligonucleotide 378 gatattatgt gtggtatttt c 21 379 59 DNA
Artificial Sequence Diagnostic Oligonucleotide 379 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg gaacttgtac attgttgca 59 380 720
DNA Artificial Sequence Diagnostic Oligonucleotide 380 agatggtaga
acctccttag agcaaaagga cacagcagtt aaatgtgaca tacctgattg 60
ttcaaaatgc aaggctctgg acattgcatt ctttgacttt tattttcctt tgagcctgtg
120 ccagtttctg tccctgctct ggtctgacct gccttctgtc ccagatctca
ctaacagcca 180 tttccctagg tcatagaaga gaacaaagtg cggcagtacg
attccatcca gaaactgctg 240 aacgagagga gcctcttccg gcaagccatc
agcccctccg acagggtgaa gctctttccc 300 caccggaact caagcaagtg
caagtctaag ccccagattg ctgctctgaa agaggagaca 360 gaagaagagg
tgcaagatac aaggctttag agagcagcat aaatgttgac atgggacatt 420
tgctcatgga attggagctc gtgggacagt cacctcatgg aattggagct cgtggaacag
480 ttacctctgc ctcagaaaac aaggatgaat taagtttttt tttaaaaaag
aaacatttgg 540 taaggggaat tgaggacact gatatgggtc ttgataaatg
gcttcctggc aatagtcaaa 600 ttgtgtgaaa ggtacttcaa atccttgaag
atttaccact tgtgttttgc aagccagatt 660 ttcctgaaaa cccttgccat
gtgctagtaa ttggaaaggc agctctaaat gtcaatcagc 720 381 56 DNA
Artificial Sequence Diagnostic Oligonucleotide 381 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg cctttgagcc tgtgcc 56 382 16 DNA
Artificial Sequence Diagnostic Oligonucleotide 382 gcttgagttc
cggtgg 16 383 16 DNA Artificial Sequence Diagnostic Oligonucleotide
383 catcagcccc tccgac 16 384 57 DNA Artificial Sequence Diagnostic
Oligonucleotide 384 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tttctgaggc agaggta 57 385 20 DNA Artificial Sequence Diagnostic
Oligonucleotide 385 gcagttaaat gtgacatacc 20 386 17 DNA Artificial
Sequence Diagnostic Oligonucleotide 386 tccttgtttt ctgaggc 17 387
62 DNA Artificial Sequence Diagnostic Oligonucleotide 387
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg ggaagccaaa tgacatcaca
60 gc 62 388 21 DNA Artificial Sequence Diagnostic Oligonucleotide
388 tgaaaaaaag tttggagaca a 21 389 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 389 cccagcgccc agagacc 17 390 62 DNA
Artificial Sequence Diagnostic Oligonucleotide 390 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg actgcttatt cctttacccc 60 aa 62 391
63 DNA Artificial Sequence Diagnostic Oligonucleotide 391
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg ccagaaaagt tgaatagtat
60 cag 63 392 19 DNA Artificial Sequence Diagnostic Oligonucleotide
392 agattgtcag cagaatcaa 19 393 16 DNA Artificial Sequence
Diagnostic Oligonucleotide 393 ataccaaatc ccttcg 16 394 59 DNA
Artificial Sequence Diagnostic Oligonucleotide 394 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg tgctttctct tctctaaat 59 395 61 DNA
Artificial Sequence Diagnostic Oligonucleotide 395 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg tggaattgtc agacatatac 60 c 61 396
17 DNA Artificial Sequence Diagnostic Oligonucleotide 396
agccaccata cttggct 17 397 56 DNA Artificial Sequence Diagnostic
Oligonucleotide 397 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tggtgttgta tggtct 56 398 17 DNA Artificial Sequence Diagnostic
Oligonucleotide 398 aacataaatc tccagaa 17 399 60 DNA Artificial
Sequence Diagnostic Oligonucleotide 399 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg tgtctctata atacttgggt 60 400 22 DNA
Artificial Sequence Diagnostic Oligonucleotide 400 atataaaaag
attccataga ac 22 401 21 DNA Artificial Sequence Diagnostic
Oligonucleotide 401 gctggcttca aagaaaaatc c 21 402 63 DNA
Artificial Sequence Diagnostic Oligonucleotide 402 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg caccagattt cgtagtcttt 60 tca 63
403 61 DNA Artificial Sequence Diagnostic Oligonucleotide 403
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg aatttctctg tttttcccct
60 t 61 404 23 DNA Artificial Sequence Diagnostic Oligonucleotide
404 agctattctt catctgcatt cca 23 405 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 405 gacactgctc ctacacc 17 406 57 DNA
Artificial Sequence Diagnostic Oligonucleotide 406 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg tcagcattta tccctta 57 407 66 DNA
Artificial Sequence Diagnostic Oligonucleotide 407 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ataatatatt tgtattttgt 60 ttgttg 66
408 20 DNA Artificial Sequence Diagnostic Oligonucleotide 408
aatttgttca ggttgttgga 20 409 18 DNA Artificial Sequence Diagnostic
Oligonucleotide 409 agctgtcaag ccgtgttc 18 410 59 DNA Artificial
Sequence Diagnostic Oligonucleotide 410 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg atctgaccca ggaaaactc 59 411 60 DNA Artificial
Sequence Diagnostic Oligonucleotide 411 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg ttgttagttt ctaggggtgg 60 412 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 412 aaggactatc
aggaaaccaa g 21 413 20 DNA Artificial Sequence Diagnostic
Oligonucleotide 413 gctaatctgg gagttgttac 20 414 62 DNA Artificial
Sequence Diagnostic Oligonucleotide 414 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg agttatgaaa ataggttgct 60 ac 62 415 58 DNA
Artificial Sequence Diagnostic Oligonucleotide 415 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg gggagaatga tgatgaag 58 416 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 416 acactgaaga
tcactgttct a 21 417 18 DNA Artificial Sequence Diagnostic
Oligonucleotide 417 tccttgccct ttttcagg 18 418 65 DNA Artificial
Sequence Diagnostic Oligonucleotide 418 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg tttaatgaca ctgaagatca 60 ctgtt 65 419 63 DNA
Artificial Sequence Diagnostic Oligonucleotide 419 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ccttgagcag ttcttaatag 60 ata 63
420 21 DNA Artificial Sequence Diagnostic Oligonucleotide 420
atgccttaac agattggata t 21 421 21 DNA Artificial Sequence
Diagnostic Oligonucleotide 421 gaaaatatcc aatctgttaa g 21 422 58
DNA Artificial Sequence Diagnostic Oligonucleotide 422 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg tgaggtggaa gtctacca 58 423 16 DNA
Artificial Sequence Diagnostic
Oligonucleotide 423 gaaaatatcc aatctg 16 424 56 DNA Artificial
Sequence Diagnostic Oligonucleotide 424 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg atgaggtgga agtcta 56 425 63 DNA Artificial
Sequence Diagnostic Oligonucleotide 425 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg agaccatgct cagatcttcc 60 att 63 426 24 DNA
Artificial Sequence Diagnostic Oligonucleotide 426 gctgccttcc
gagtcagttt cagt 24 427 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 427 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
actgaaactg actcggaagg 60 428 26 DNA Artificial Sequence Diagnostic
Oligonucleotide 428 atggtacatt acctgtattt tgttta 26 429 26 DNA
Artificial Sequence Diagnostic Oligonucleotide 429 ctgtacaaac
atggtatgac tctctt 26 430 65 DNA Artificial Sequence Diagnostic
Oligonucleotide 430 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gtgaaggaaa tttctttttc 60 tatct 65 431 24 DNA Artificial Sequence
Diagnostic Oligonucleotide 431 agaccatgct cagatcttcc attc 24 432 62
DNA Artificial Sequence Diagnostic Oligonucleotide 432 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg tgccttccga gtcagtttca 60 gt 62 433
62 DNA Artificial Sequence Diagnostic Oligonucleotide 433
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg gcacaatgag agtataaagt
60 ag 62 434 21 DNA Artificial Sequence Diagnostic Oligonucleotide
434 ccatcactac ttctgtagtc g 21 435 27 DNA Artificial Sequence
Diagnostic Oligonucleotide 435 ctctctttta taaataggat ttcttac 27 436
65 DNA Artificial Sequence Diagnostic Oligonucleotide 436
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg ttccagttct accagttata
60 tcatc 65 437 67 DNA Artificial Sequence Diagnostic
Oligonucleotide 437 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ctctctttta taaataggat 60 ttcttac 67 438 25 DNA Artificial Sequence
Diagnostic Oligonucleotide 438 ttccagttct accagttata tcatc 25 439
65 DNA Artificial Sequence Diagnostic Oligonucleotide 439
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg acaatagaaa aacttctaat
60 ggtga 65 440 22 DNA Artificial Sequence Diagnostic
Oligonucleotide 440 aaaaaagaga catggacacc aa 22 441 18 DNA
Artificial Sequence Diagnostic Oligonucleotide 441 cctgagcgtg
atttgata 18 442 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 442 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
atgtagacta accgattgaa 60 443 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 443 gggagaactg gagcct 16 444 58 DNA Artificial
Sequence Diagnostic Oligonucleotide 444 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg aaccgattga atatggag 58 445 61 DNA Artificial
Sequence Diagnostic Oligonucleotide 445 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg gatatatgat tacattagaa 60 g 61 446 17 DNA
Artificial Sequence Diagnostic Oligonucleotide 446 accttctcca
agaacta 17 447 18 DNA Artificial Sequence Diagnostic
Oligonucleotide 447 ataggacatc tccaagtt 18 448 59 DNA Artificial
Sequence Diagnostic Oligonucleotide 448 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg gcaatagaga aatgtctgt 59 449 63 DNA Artificial
Sequence Diagnostic Oligonucleotide 449 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg gtgaactgtt taaggcaaat 60 cat 63 450 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 450 tgatgggaca
gtctgtcttt c 21 451 25 DNA Artificial Sequence Diagnostic
Oligonucleotide 451 aatacgagac atattgcaat aaagt 25 452 62 DNA
Artificial Sequence Diagnostic Oligonucleotide 452 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ctggctgtag attttggagt 60 tc 62 453
25 DNA Artificial Sequence Diagnostic Oligonucleotide 453
aatacgagac atattgcaat aaagt 25 454 21 DNA Artificial Sequence
Diagnostic Oligonucleotide 454 ctggctgtag attttggagt t 21 455 17
DNA Artificial Sequence Diagnostic Oligonucleotide 455 aggtagcagc
tattttt 17 456 57 DNA Artificial Sequence Diagnostic
Oligonucleotide 456 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ggacagcctt ctctcta 57 457 25 DNA Artificial Sequence Diagnostic
Oligonucleotide 457 cacttctaaa atggaacatt taaag 25 458 58 DNA
Artificial Sequence Diagnostic Oligonucleotide 458 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg gcagtatcgc ctctccct 58 459 61 DNA
Artificial Sequence Diagnostic Oligonucleotide 459 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg atgggacatt ttcagaactc 60 c 61 460
19 DNA Artificial Sequence Diagnostic Oligonucleotide 460
cctcttcgat gccattcat 19 461 63 DNA Artificial Sequence Diagnostic
Oligonucleotide 461 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
caatccaatc aactctatac 60 gaa 63 462 18 DNA Artificial Sequence
Diagnostic Oligonucleotide 462 ctgatcacgc tgatgcga 18 463 60 DNA
Artificial Sequence Diagnostic Oligonucleotide 463 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg tgatgagcct ttagagagaa 60 464 19
DNA Artificial Sequence Diagnostic Oligonucleotide 464 ccagttcagt
caagtttgc 19 465 54 DNA Artificial Sequence Diagnostic
Oligonucleotide 465 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
cagcgtgatc agca 54 466 18 DNA Artificial Sequence Diagnostic
Oligonucleotide 466 tttgttacat gctacata 18 467 58 DNA Artificial
Sequence Diagnostic Oligonucleotide 467 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg catcagcgtg atcagcac 58 468 26 DNA Artificial
Sequence Diagnostic Oligonucleotide 468 tagtaaattt tgttacatgc
tacata 26 469 62 DNA Artificial Sequence Diagnostic Oligonucleotide
469 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg ttcatatatt
aaaaataaaa 60 cc 62 470 21 DNA Artificial Sequence Diagnostic
Oligonucleotide 470 taatatatcg aaggtatgtg t 21 471 22 DNA
Artificial Sequence Diagnostic Oligonucleotide 471 gagcatacca
gcagtgacta ca 22 472 69 DNA Artificial Sequence Diagnostic
Oligonucleotide 472 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gtaatacttt acaatagaac 60 attcttacc 69 473 20 DNA Artificial
Sequence Diagnostic Oligonucleotide 473 accagcagtg actacatgga 20
474 68 DNA Artificial Sequence Diagnostic Oligonucleotide 474
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg atatttatgt gtgtgcatat
60 atatgtat 68 475 57 DNA Artificial Sequence Diagnostic
Oligonucleotide 475 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gtgtaccttg atattgg 57 476 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 476 ctcactttcc aaggag 16 477 15 DNA Artificial
Sequence Diagnostic Oligonucleotide 477 gctgtggctc cttgg 15 478 57
DNA Artificial Sequence Diagnostic Oligonucleotide 478 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg actacagccc gaactcc 57 479 19 DNA
Artificial Sequence Diagnostic Oligonucleotide 479 gtggctgctt
ctttggttg 19 480 69 DNA Artificial Sequence Diagnostic
Oligonucleotide 480 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tacaaacata gtggattaca 60 atatttaat 69 481 66 DNA Artificial
Sequence Diagnostic Oligonucleotide 481 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg catgtattgg aaattcagta 60 agtaac 66 482 24 DNA
Artificial Sequence Diagnostic Oligonucleotide 482 ttcgacactg
tgattagagt atgc 24 483 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 483 gtgggagtag ccgaca 16 484 57 DNA Artificial
Sequence Diagnostic Oligonucleotide 484 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg caggccctat tgatggt 57 485 16 DNA Artificial
Sequence Diagnostic Oligonucleotide 485 cgtgggagta gccgac 16 486 58
DNA Artificial Sequence Diagnostic Oligonucleotide 486 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg cattagaaaa ccaacaaa 58 487 57 DNA
Artificial Sequence Diagnostic Oligonucleotide 487 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg ctgaatgcgt cttactg 57 488 17 DNA
Artificial Sequence Diagnostic Oligonucleotide 488 catccaaaat
tgctata 17 489 66 DNA Artificial Sequence Diagnostic
Oligonucleotide 489 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
cgtctactgt gatccaaact 60 tagtat 66 490 24 DNA Artificial Sequence
Diagnostic Oligonucleotide 490 catacctgga tgaagtcaaa tatg 24 491 23
DNA Artificial Sequence Diagnostic Oligonucleotide 491 ttgaggaatt
tgtcatcttg tat 23 492 63 DNA Artificial Sequence Diagnostic
Oligonucleotide 492 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
caaaatcaca tttgcttttg 60 tta 63 493 58 DNA Artificial Sequence
Diagnostic Oligonucleotide 493 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg aaagaaataa atcactga 58 494 16 DNA Artificial Sequence
Diagnostic Oligonucleotide 494 gtaaaactgc gacaac 16 495 26 DNA
Artificial Sequence Diagnostic Oligonucleotide 495 ccaacatgtt
ttctttgatc ttacag 26 496 67 DNA Artificial Sequence Diagnostic
Oligonucleotide 496 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
agaatctcaa atagctctta 60 tagcttt 67 497 62 DNA Artificial Sequence
Diagnostic Oligonucleotide 497 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg ttaaccaatg acatttgtga 60 ta 62 498 21 DNA Artificial
Sequence Diagnostic Oligonucleotide 498 gtgtccatag tccttttaag c 21
499 61 DNA Artificial Sequence Diagnostic Oligonucleotide 499
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg atatttcaca ggcaggagtc
60 c 61 500 25 DNA Artificial Sequence Diagnostic Oligonucleotide
500 aaaatcattt ctattctcat ttgga 25 501 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 501 acttcgtgcc ttcggac 17 502 62 DNA
Artificial Sequence Diagnostic Oligonucleotide 502 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg cagcaatgaa gaagatgaca 60 aa 62 503
17 DNA Artificial Sequence Diagnostic Oligonucleotide 503
ctggttccaa atgagaa 17 504 59 DNA Artificial Sequence Diagnostic
Oligonucleotide 504 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
taacctatag aatgcagca 59 505 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 505 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ttaatgtgat atgtgcccta 60 506 21 DNA Artificial Sequence Diagnostic
Oligonucleotide 506 agatgataag acttaccaag c 21 507 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 507 gagaaggaga
aggaagagtt g 21 508 63 DNA Artificial Sequence Diagnostic
Oligonucleotide 508 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
cttcctcatg ctattactca 60 tac 63 509 61 DNA Artificial Sequence
Diagnostic Oligonucleotide 509 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg aagttatttt ttaggaagca 60 t 61 510 19 DNA Artificial
Sequence Diagnostic Oligonucleotide 510 gaacttaaag actcggctc 19 511
61 DNA Artificial Sequence Diagnostic Oligonucleotide 511
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg gaaattgtct gccattctta
60 a 61 512 20 DNA Artificial Sequence Diagnostic Oligonucleotide
512 gagttggcca ttcttgtatg 20 513 56 DNA Artificial Sequence
Diagnostic Oligonucleotide 513 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg tgtgagccga gtcttt 56 514 16 DNA Artificial Sequence
Diagnostic Oligonucleotide 514 atggcatttc cacctt 16 515 61 DNA
Artificial Sequence Diagnostic Oligonucleotide 515 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg tgttattttt atttcagatg 60 c 61 516
19 DNA Artificial Sequence Diagnostic Oligonucleotide 516
taatatggca tttccacct 19 517 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 517 cgtgaagaaa gatgac 16 518 59 DNA Artificial
Sequence Diagnostic Oligonucleotide 518 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg taatgttaca aatagattc 59 519 21 DNA Artificial
Sequence Diagnostic Oligonucleotide 519 cacacgtgaa gaaagatgac a 21
520 66 DNA Artificial Sequence Diagnostic Oligonucleotide 520
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg taatgttaca aatagattct
60 gctaac 66 521 56 DNA Artificial Sequence Diagnostic
Oligonucleotide 521 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
cttgatttct ggagac 56 522 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 522 ctagctgtaa ttgcat 16 523 61 DNA Artificial
Sequence Diagnostic Oligonucleotide 523 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg gaattatgtt tatggcatgg 60 t 61 524 25 DNA
Artificial Sequence Diagnostic Oligonucleotide 524 gagtacaagt
atcaaatagc agtaa 25 525 59 DNA Artificial Sequence Diagnostic
Oligonucleotide 525 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ctgaattatg tttatggca 59 526 18 DNA Artificial Sequence Diagnostic
Oligonucleotide 526 ccttttttct ggctaagt 18 527 61 DNA Artificial
Sequence Diagnostic Oligonucleotide 527 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg gatggtaagt acatgggtgt 60 t 61 528 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 528 actccactgt
tcatagggat c 21 529 60 DNA Artificial Sequence Diagnostic
Oligonucleotide 529 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
agttattcat actttcttct 60 530 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 530 agccttacct catctg 16 531 56 DNA Artificial
Sequence Diagnostic Oligonucleotide 531 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg tttctggaac atttag 56 532 18 DNA Artificial
Sequence Diagnostic Oligonucleotide 532 gaatgatgtc agctatat 18 533
63 DNA Artificial Sequence Diagnostic Oligonucleotide 533
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tttctggaac atttagaaaa 60 aac 63 534 19 DNA Artificial Sequence
Diagnostic Oligonucleotide 534 tcagttaggg gtaggtcca 19 535 58 DNA
Artificial Sequence Diagnostic Oligonucleotide 535 cgcccgccgc
gccccgcgcc cgccccgccg cccccgcccg tgagctgtca aggttgta 58 536 19 DNA
Artificial Sequence Diagnostic Oligonucleotide 536 caggaaactg
ttctatcac 19 537 64 DNA Artificial Sequence Diagnostic
Oligonucleotide 537 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gaatgtcaac tgcttgagtg 60 tttt 64 538 27 DNA Artificial Sequence
Diagnostic Oligonucleotide 538 aagtaacaga acatctgaaa ctcacac 27 539
16 DNA Artificial Sequence Diagnostic Oligonucleotide 539
cttgcgcttg atgaac 16 540 63 DNA Artificial Sequence Diagnostic
Oligonucleotide 540 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
tgggcaatta tttcatatct 60 tgg 63 541 58 DNA Artificial Sequence
Diagnostic Oligonucleotide 541 cgcccgccgc gccccgcgcc cgccccgccg
cccccgcccg tatcaaggta aatacaga 58 542 17 DNA Artificial Sequence
Diagnostic Oligonucleotide 542 gcttctatcc tgtgttc 17 543 21 DNA
Artificial Sequence Diagnostic Oligonucleotide 543 gatattatgt
gtggtatttt c 21 544 59 DNA Artificial Sequence Diagnostic
Oligonucleotide 544 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
gaacttgtac attgttgca 59 545 67 DNA Artificial Sequence Diagnostic
Oligonucleotide 545 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
catattatca aggtaaatac 60 agatcat 67 546 25 DNA Artificial Sequence
Diagnostic Oligonucleotide 546 ggaactatca catgtgagat tgtta 25 547
56 DNA Artificial Sequence Diagnostic Oligonucleotide 547
cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg cctttgagcc tgtgcc 56
548 16 DNA Artificial Sequence Diagnostic Oligonucleotide 548
gcttgagttc cggtgg 16 549 16 DNA Artificial Sequence Diagnostic
Oligonucleotide 549 catcagcccc tccgac 16 550 57 DNA Artificial
Sequence Diagnostic Oligonucleotide 550 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg tttctgaggc agaggta 57 551 58 DNA Artificial
Sequence Diagnostic Oligonucleotide 551 cgcccgccgc gccccgcgcc
cgccccgccg cccccgcccg cattgcattc tttgactt 58 552 16 DNA Artificial
Sequence Diagnostic Oligonucleotide 552 aagagctcac cctgtc 16 553 17
DNA Artificial Sequence Diagnostic Oligonucleotide 553 gcagtacgat
tccatcc 17 554 56 DNA Artificial Sequence Diagnostic
Oligonucleotide 554 cgcccgccgc gccccgcgcc cgccccgccg cccccgcccg
ccttgttttc tgaggc 56
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